Rnai-mediated inhibition of ocular hypertension targets

ABSTRACT

RNA interference is provided for inhibition of ocular hypertension target mRNA expression for lowering elevated intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Ocular hypertension targets include carbonic anhydrase II, IV, and XII; β1- and β2 adrenergic receptors; acetylcholinesterase; Na + /K + -ATPase; and Na—K-2Cl cotransporter. Ocular hypertension is treated by administering interfering RNAs of the present invention.

The present application is a divisional of U.S. patent application Ser. No. 11/344,702 filed Feb. 1, 2006, which claims the benefit of co-pending U.S. Provisional Patent Applications having Ser. Nos. 60/648,926 filed Feb. 1, 2005, and 60/753,364 filed Dec. 22, 2005, the texts of which are specifically incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of interfering RNA compositions for inhibition of expression of ocular hypertension targets in glaucoma, particularly for primary open angle glaucoma.

BACKGROUND OF THE INVENTION

Glaucoma is a heterogeneous group of optic neuropathies that share certain clinical features. The loss of vision in glaucoma is due to the selective death of retinal ganglion cells in the neural retina that is clinically diagnosed by characteristic changes in the visual field, nerve fiber layer defects, and a progressive cupping of the optic nerve head (ONH). One of the main risk factors for the development of glaucoma is the presence of ocular hypertension (elevated intraocular pressure, IOP). An adequate intraocular pressure is needed to maintain the shape of the eye and to provide a pressure gradient to allow for the flow of aqueous humor to the avascular cornea and lens. IOP levels may also be involved in the pathogenesis of normal tension glaucoma (NTG), as evidenced by patients benefiting from IOP lowering medications. Once adjustments for central corneal thickness are made to IOP readings in NTG patients, many of these patients may be found to be ocular hypertensive.

The elevated IOP associated with glaucoma is due to elevated aqueous humor outflow resistance in the trabecular meshwork (TM), a small specialized tissue located in the iris-corneal angle of the ocular anterior chamber. Glaucomatous changes to the TM include a loss in TM cells and the deposition and accumulation of extracellular debris including proteinaceous plaque-like material. In addition, there are also changes that occur in the glaucomatous ONH. In glaucomatous eyes, there are morphological and mobility changes in ONH glial cells. In response to elevated IOP and/or transient ischemic insults, there is a change in the composition of the ONH extracellular matrix and alterations in the glial cell and retinal ganglion cell axon morphologies.

Primary glaucomas result from disturbances in the flow of intraocular fluid that has an anatomical or physiological basis. Secondary glaucomas occur as a result of injury or trauma to the eye or a preexisting disease. Primary open angle glaucoma (POAG), also known as chronic or simple glaucoma, represents ninety percent of all primary glaucomas. POAG is characterized by the degeneration of the trabecular meshwork, resulting in abnormally high resistance to fluid drainage from the eye. A consequence of such resistance is an increase in the IOP that is required to drive the fluid normally produced by the eye across the increased resistance.

Current anti-glaucoma therapies include lowering IOP by the use of suppressants of aqueous humor formation or agents that enhance uveoscleral outflow, laser trabeculoplasty, or trabeculectomy, which is a filtration surgery to improve drainage. Pharmaceutical anti-glaucoma approaches have exhibited various undesirable side effects. For example, miotics such as pilocarpine can cause blurring of vision and other negative visual side effects. Systemically administered carbonic anhydrase inhibitors (CAIs) can also cause nausea, dyspepsia, fatigue, and metabolic acidosis. Further, certain beta-blockers have increasingly become associated with serious pulmonary side effects attributable to their effects on beta-2 receptors in pulmonary tissue. Sympathomimetics cause tachycardia, arrhythmia and hypertension. Such negative side effects may lead to decreased patient compliance or to termination of therapy. In addition, the efficacy of current IOP lowering therapies is relatively short-lived requiring repeated dosing during each day and, in some cases, the efficacy decreases with time.

In view of the importance of ocular hypertension in glaucoma, and the inadequacies of prior methods of treatment, it would be desirable to have an improved method of treating ocular hypertension that would address the underlying causes of its progression.

SUMMARY OF THE INVENTION

The present invention is directed to interfering RNAs that silence ocular hypertension target mRNA expression, thus lowering intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Ocular hypertension targets include carbonic anhydrase II, IV, and XII; β1- and β2 adrenergic receptors; acetylcholinesterase; Na⁺/K⁺-ATPase; and Na—K-2Cl cotransporter. The interfering RNAs of the invention are useful for treating patients with open-angle glaucoma or ocular hypertension.

An embodiment of the present invention provides a method of attenuating expression of an ocular hypertension target mRNA such as carbonic anhydrase II, IV, or XII; β1- or β2 adrenergic receptors; acetylcholinesterase; Na⁺/K⁺-ATPase; or Na—K-2Cl cotransporter mRNA in a subject. The method comprises administering to the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier. Administration is to the eye of the subject for attenuating expression of an ocular hypertension target in a human.

In one embodiment of the invention, the interfering RNA comprises a sense nucleotide strand, an antisense nucleotide strand and a region of at least near-perfect contiguous complementarity of at least 19 nucleotides. Further, the antisense strand hybridizes under physiological conditions to a portion of an mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134 which are sense cDNA sequences encoding carbonic anhydrase II and IV; β1- and β2 adrenergic receptors; acetylcholinesterase (ACHE) variant E4-E5; Na⁺/K⁺-ATPase α2 polypeptide; Na—K-2Cl cotransporter NKCC2 (SLC12A1), carbonic anhydrase XII variant 1, acetylcholinesterase variant E4-E6, Na⁺/K⁺-ATPase α1 polypeptide variant 1 and variant 2, Na⁺/K⁺-ATPase α3 polypeptide, Na⁺/K⁺-ATPase α4 polypeptide variant 1 and variant 2, Na⁺/K⁺-ATPase β1 polypeptide variant 1 and 2, Na⁺/K⁺-ATPase β2 polypeptide, Na⁺/K⁺-ATPase β3 polypeptide, Na—K-2Cl cotransporter NKCC1 (SLC12A2), and carbonic anhydrase XII variant 2, respectively. The antisense strand has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, respectively. The administration of such a composition attenuates the expression of an ocular hypertension target mRNA of the subject.

In one embodiment, the ocular hypertension target mRNA encodes carbonic anhydrase II, IV or XII, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:101, or SEQ ID NO:134 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:101, or SEQ ID NO:134, respectively.

In another embodiment, the ocular hypertension target mRNA encodes a β1- or β2-adrenergic receptor, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:3 or SEQ ID NO:4 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:3 or SEQ ID NO:4, respectively.

In a further embodiment, the ocular hypertension target mRNA encodes an acetylcholinesterase, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:5 or SEQ ID NO:123 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:5 or SEQ ID NO:123, respectively.

In yet another embodiment, the ocular hypertension target mRNA encodes a subunit of Na⁺/K⁺-ATPase, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:6, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, or SEQ ID NO:132 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:6, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, or SEQ ID NO:132, respectively.

In a further embodiment, the ocular hypertension target mRNA encodes a Na—K-2Cl cotransporter, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:7 or SEQ ID NO:133 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:7 or SEQ ID NO:133, respectively.

In one embodiment of the invention, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:1 comprising nucleotide 232, 527, 721, 728, 809, 810, 855, 856, 921, 1139, 506, 547, 548, 740, 911, 1009, 1140, 1149, 1150, 1151, 1188, 1194, 1195, 1223, 1239, 1456, 1457, 1458, 100, 158, 166, 247, 286, 318, 322, 328, 371, 412, 482, 504, 505, 541, 734, 772, 777, 814, 972, 998, 1232, 317, or 401.

In another embodiment of the invention, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:2 comprising nucleotide 213, 252, 258, 266, 399, 457, 463, 490, 595, 1064, 109, 112, 125, 126, 150, 261, 265, 280, 398, 453, 459, 462, 467, 492, 534, 785, 801, 825, 827, 876, 1003, or 1012.

In a further embodiment of the invention, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:101 comprising nucleotide 191, 239, 274, 275, 341, 389, 412, 413, 423, 687, 689, 695, 710, 791, 792, 794, 983, 993, 994, 995, 691, 1039, 1568, 2326, 2332, 2425, 2433, 2844, 2845, 2880, 2884, 2891, 2954, 2955, 2956, 2957, 2964, 2965, 3006, 3007, 3012, or 3026.

In another embodiment, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:134 comprising nucleotide 687, 1535, 2293, 2299, 2392, 2400, 2811, 2812, 2847, 2851, 2858, 2921, 2922, 2923, 2924, 2931, 2932, 2973, 2974, 2979, or 2993.

Another embodiment of the invention provides an interfering RNA designed to target an mRNA corresponding to SEQ ID NO:3 comprising nucleotide 468, 523, 799, 1563, 1565, 1569, 1593, 1613, 1614, 1626, 310, 322, 726, 769, 772, 801, 802, 1501, 1576, 1577, 1579, 1580, 1581, 1586, 1590, 1592, 1594, 1615, 1616, 1632, 1633, or 1654.

A further embodiment of the invention provides an interfering RNA designed to target an mRNA corresponding to SEQ ID NO:4 comprising nucleotide 329, 375, 1031, 1046, 1149, 1163, 1371, 1401, 1426, 1880, 283, 607, 608, 609, 619, 623, 722, 857, 1037, 1091, 1115, 1124, 1136, 1137, 1151, 1164, 1393, 1394, 1395, 1406, 1407, 1427, 1428, 1429, 1442, 1725, 1726, 1756, 1757, 1758, 1767, 1790, 1791, 1792, 1793, 1803, 1861, 1869, 1971, 1972, or 1979.

In another method of the invention, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:123 comprising nucleotide 1875, 1890, 1891, 2011, 2012, 2133, or 2134.

Another embodiment of the invention provides an interfering RNA designed to target an mRNA corresponding to SEQ ID NO:5 comprising nucleotide 366, 370, 384, 385, 525, 588, 768, 1045, 1046, 1061, 1090, 1232, 1314, 1316, 1460, 1461, 1462, 1528, 1607, 1705, 1713, 382, 393, 397, 622, 1131, 1459, 1530, 2251, 2885, 2886, 386, 1231, 1315, 2047, 2049, 2053, 2055, 2057, 2125, 2126, 2127, 2250, 2253, 2258, 2260, 2318, 2395, 2397, 2404, 2405, 2643, 2645, or 2887.

In a further embodiment, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:124 comprising nucleotide 2208, 2275, 2307, 2526, 2538, 2592, 2628, 2979, 2985, 3093, 3474, 3504, 3505, 3506, 3518, 343, 442, 700, 707, 811, 907, 1059, 1363, 1594, 1662, 1758, 1760, 1896, 2037, or 2147.

In yet another embodiment, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:125 comprising nucleotide 436, 441, 443, 552, 617, 701, 702, 832, 2204, 2291, or 2495.

A further embodiment of the present invention provides an interfering RNA designed to target an mRNA corresponding to SEQ ID NO:6 comprising nucleotide 471, 1990, 3080, 3797, 4037, 4093, 4225, 4323, 5213, 5285, 214, 467, 470, 472, 473, 632, 825, 946, 1693, 1767, 1768, 2157, 2263, 2589, 2590, 2765, 2988, 3094, 3144, 3145, 3344, 3345, 3418, 3666, 3828, 3850, 4040, 4041, 4061, 4882, 4894, 4900, 5040, 5114, 5115, 5128, 5129, 5253, 5296, 5375, 5384, or 5385.

In another embodiment of the invention, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:126 comprising nucleotide 240, 272, 362, 1836, 1851, 2103, 2137, 2138, 2139, 2157, 2158, 2160, 2425, 2580, 2601, 2646, 2650, 2794, 2803, 3116, 3124, 3126, 3129, or 3377.

In yet another embodiment of the invention, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:127 comprising nucleotide 113, 612, 702, 833, 1101, 1732, 1733, 1836, 2070, 2071, 2143, 2328, 2475, 2861, 2862, 2952, 3203, 3281, 3377, 3379, 3470, 3471, 3554, 3614, 3615, 3616, 3617, 3625, 3626, 3642, 3646, 3647, 3653, 3655, 3797, 3801, 3803, 3809 or 3810.

In another embodiment, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:128 comprising nucleotide 126, 251, 252, 253, 331, 427, 429, 520, 521, 530, 601, 602, 603, 604, 664, 665, 666, 667, 675, 676, 692, 696, 697, 702, 703, 705, 707, 847, 851, 853, 859, or 860.

In yet another embodiment, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:129 comprising nucleotide 1096, 1099, 1130, 1131, 1167, 1299, 1441, 1450, 1451, 1452, 1564, 1746, 1750, 1751, 1752, 1795, 203, 204, 214, 222, 224, 225, 226, 380, 525, 591, 612, 613, 615, 635, 636, 663, 664, 669, 699, 765, 790, 839, 840, 841, 900, 909, 933, or 947.

In another embodiment, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:130 comprising nucleotide 1063, 1102, 1106, 1107, 1108, 1109, 1111, or 1151.

In another embodiment, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:131 comprising nucleotide 653, 654, 771, 773, 841, 849, 853, 917, 918, 926, 927, 931, 981, 983, 984, 996, 998, 1022, 1023, 1160, 1214, 1355, 1356, 1381, 1394, 1425, 1474, 1550, 1620, 1707, 1740, 1753, 1825, 1956, 1965, 2598, 2599, 2608, 2828, 2829, 2888, 3012, or 3251.

In another embodiment of the invention, an interfering RNA is designed to target an mRNA corresponding to SEQ ID NO:132 comprising nucleotide 292, 434, 438, 457, 459, 488, 490, 498, 499, 592, 639, 723, 774, 775, 788, 857, 858, 910, 911, 930, 931, 932, 1009, 1010, 1023, 1024, 1111, 1146, 1147, 1220, 1246, 1321, 1325, 1326, 1327, 1331, 1437, 1548, 1571, 1785, 1786, or 1787.

Another embodiment of the present invention provides an interfering RNA designed to target an mRNA corresponding to SEQ ID NO:7 comprising nucleotide 675, 974, 1373, 1780, 2102, 2151, 2315, 2542, 2609, 3197, 67, 71, 73, 353, 405, 864, 911, 912, 913, 1409, 1748, 1811, 1935, 1937, 1993, 2012, 2346, 2388, 2437, 2586, 3007, 3008, 3022, 3130, 3210, 3237, or 3271.

Another embodiment of the present invention provides an interfering RNA designed to target an mRNA corresponding to SEQ ID NO:133 comprising nucleotide 748, 749, 753, 1119, 1169, 1499, 1509, 1820, 2081, 2118, 2147, 2615, 2644, 2659, 2663, 2671, 2672, 2793, 2812, 2914, 2948, 3044, 3334, 3391, 3480, 3520, 3549, 3639, 3840, 3941, 3944, 4001, 4995, 4997, 5141, 5143, 5249, 5375, 5834, 5852, 5981, or 6678.

The present invention further provides for administering a second interfering RNA to a subject in addition to a first interfering RNA. The method comprises administering to the subject a second interfering RNA having a length of 19 to 49 nucleotides and comprising a sense nucleotide strand, an antisense nucleotide strand, and a region of at least near-perfect complementarity of at least 19 nucleotides; wherein the antisense strand of the second interfering RNA hybridizes under physiological conditions to a second portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, and the antisense strand has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the second hybridizing portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, respectively. The second interfering RNA may target the same mRNA as the first interfering RNA or may target a different mRNA. Further, a third, fourth, or fifth, etc. interfering RNA may be administered in a similar manner.

A further embodiment of the invention is a method of treating ocular hypertension in a subject in need thereof. The method comprises administering to the eye of the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising a sense nucleotide strand, an antisense nucleotide strand, and a region of at least near-perfect contiguous complementarity of at least 19 nucleotides. The antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, respectively. The ocular hypertension is treated thereby.

Another embodiment of the invention is a method of attenuating expression of an ocular hypertension target mRNA in a subject comprising administering to the subject a composition comprising an effective amount of single-stranded interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier. For attenuating expression of an ocular hypertension target, the single-stranded interfering RNA hybridizes under physiological conditions to a portion of mRNA corresponding to the sequence identifiers and nucleotide positions cited supra for antisense strands.

Another embodiment of the invention is a method of attenuating expression of an ocular hypertension target mRNA in a subject, comprising administering to the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, where the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:8, SEQ ID NO:14-SEQ ID NO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ ID NO:135-SEQ ID NO:717, SEQ ID NO:720, and SEQ ID NO:721, as follows.

When the ocular hypertension target mRNA encodes carbonic anhydrase mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:8, SEQ ID NO:14-SEQ ID NO:32, SEQ ID NO:83-SEQ ID NO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ ID NO:135-SEQ ID NO:219, SEQ ID NO:720, and SEQ ID NO:721.

When the ocular hypertension target mRNA encodes a β-adrenergic receptor mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:33-SEQ ID NO:52, and SEQ ID NO:220-SEQ ID NO:282.

When the ocular hypertension target mRNA encodes ACHE mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:53-SEQ ID NO:62 and SEQ ID NO:283-333.

When the ocular hypertension target mRNA encodes ATP1A1 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:334-SEQ ID NO:374.

When the ocular hypertension target mRNA encodes ATP1A2 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:63-SEQ ID NO:72 and SEQ ID NO:375-SEQ ID NO:416.

When the ocular hypertension target mRNA encodes ATP1A3 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:417-SEQ ID NO:440.

When the ocular hypertension target mRNA encodes ATP1A4 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:441-SEQ ID NO:511.

When the ocular hypertension target mRNA encodes ATP1B1 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:512-SEQ ID NO:563.

When the ocular hypertension target mRNA encodes ATP1B2 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:564-SEQ ID NO:606.

When the ocular hypertension target mRNA encodes ATP1B3 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:607-SEQ ID NO:648.

When the ocular hypertension target mRNA encodes SLC12A1 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:73-SEQ ID NO:82 and SEQ ID NO:649-SEQ ID NO:675.

When the ocular hypertension target mRNA encodes SLC12A2 mRNA, the interfering RNA comprises a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:676-SEQ ID NO:717.

In a further embodiment of the present invention, the region of contiguous nucleotides is a region of at least 14 contiguous nucleotides having at least 85% sequence complementarity to, or at least 85% sequence identity with, the penultimate 14 nucleotides of the 3′ end of the sequence of the sequence identifier. In yet another embodiment of the invention, the region of contiguous nucleotides is a region of at least 15, 16, 17, or 18 contiguous nucleotides having at least 80% sequence complementarity to, or at least 80% sequence identity with, the penultimate 15, 16, 17, or 18 nucleotides, respectively, of the 3′ end of the sequence of the sequence identifier.

A composition comprising interfering RNA having a length of 19 to 49 nucleotides and having a nucleotide sequence of any one of SEQ ID NO's: 8, SEQ ID NO:14-SEQ ID NO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ ID NO:135-SEQ ID NO:717, SEQ ID NO:720, and SEQ ID NO:721, or a complement thereof, and a pharmaceutically acceptable carrier is an embodiment of the present invention. In one embodiment, the interfering RNA is isolated. The term “isolated” means that the interfering RNA is free of its total natural mileau.

Another embodiment of the invention is a method of treating ocular hypertension in a subject in need thereof, the method comprising administering to an eye of the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of SEQ ID NO:8, SEQ ID NO:14-SEQ ID NO:100, SEQ ID NO:102-SEQ ID NO:122, SEQ ID NO:135-SEQ ID NO:717, SEQ ID NO:720, and SEQ ID NO:721, wherein the ocular hypertension is treated thereby.

A method of attenuating expression of an ocular hypertension target mRNA first variant without attenuating expression of an ocular hypertension target mRNA second variant in a subject is a further embodiment of the invention. The method comprises administering to the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of the first variant, wherein the expression of the first variant mRNA is attenuated without attenuating expression of the second variant mRNA, and wherein the first variant target mRNA is SEQ ID NO:101, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:127, or SEQ ID NO:129, and the second variant target mRNA is SEQ ID NO:134, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, or SEQ ID NO:130, respectively.

In a further embodiment of the above-cited method, the first variant target mRNA is SEQ ID NO:134, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:128, or SEQ ID NO:130, and the second variant target mRNA is SEQ ID NO:101, SEQ ID NO:5, SEQ ID NO:124, SEQ ID NO:127, or SEQ ID NO:129, respectively.

Use of any of the embodiments as described herein in the preparation of a medicament for attenuating expression of an ocular hypertension mRNA is also an embodiment of the present invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 provides a western blot, probed with antibodies against CA2 and actin, of HeLa cells transfected with CA2 siRNAs #1, #3, #4, and #5; a non-targeting control siRNA; and a buffer control (-siRNA). The siRNAs were at a concentration of 100 nM or 1 nM. The arrows indicate the positions of the ˜30-kDa CA2 protein and 42-kDa actin protein bands.

DETAILED DESCRIPTION OF THE INVENTION

RNA interference (RNAi) is a process by which double-stranded RNA (dsRNA) is used to silence gene expression. While not wanting to be bound by theory, RNAi begins with the cleavage of longer dsRNAs into small interfering RNAs (siRNAs) by an RNaseIII-like enzyme, dicer. SiRNAs are dsRNAs that are usually about 19 to 28 nucleotides, or 20 to 25 nucleotides, or 21 to 22 nucleotides in length and often contain 2-nucleotide 3′ overhangs, and 5′ phosphate and 3′ hydroxyl termini. One strand of the siRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). RISC uses this siRNA strand to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, and then cleaves these target mRNAs or inhibits their translation. Therefore, the siRNA strand that is incorporated into RISC is known as the guide strand or the antisense strand. The other siRNA strand, known as the passenger strand or the sense strand, is eliminated from the siRNA and is at least partially homologous to the target mRNA. Those of skill in the art will recognize that, in principle, either strand of an siRNA can be incorporated into RISC and function as a guide strand. However, siRNA design (e.g., decreased siRNA duplex stability at the 5′ end of the antisense strand) can favor incorporation of the antisense strand into RISC.

RISC-mediated cleavage of mRNAs having a sequence at least partially complementary to the guide strand leads to a decrease in the steady state level of that mRNA and of the corresponding protein encoded by this mRNA. Alternatively, RISC can also decrease expression of the corresponding protein via translational repression without cleavage of the target mRNA. Other RNA molecules and RNA-like molecules can also interact with RISC and silence gene expression. Examples of other RNA molecules that can interact with RISC include short hairpin RNAs (shRNAs), single-stranded siRNAs, microRNAs (miRNAs), and dicer-substrate 27-mer duplexes. The term “siRNA” as used herein refers to a double-stranded interfering RNA unless otherwise noted. Examples of RNA-like molecules that can interact with RISC include RNA molecules containing one or more chemically modified nucleotides, one or more deoxyribonucleotides, and/or one or more non-phosphodiester linkages. For purposes of the present discussion, all RNA or RNA-like molecules that can interact with RISC and participate in RISC-mediated changes in gene expression will be referred to as “interfering RNAs.” SiRNAs, shRNAs, miRNAs, and dicer-substrate 27-mer duplexes are, therefore, subsets of “interfering RNAs.”

Interfering RNA of embodiments of the invention appear to act in a catalytic manner for cleavage of target mRNA, i.e., interfering RNA is able to effect inhibition of target mRNA in substoichiometric amounts. As compared to antisense therapies, significantly less interfering RNA is required to provide a therapeutic effect under such cleavage conditions.

The present invention relates to the use of interfering RNA to inhibit the expression of ocular hypertension target mRNA, thus lowering intraocular pressure in patients with open-angle glaucoma or ocular hypertension. Ocular hypertension targets include carbonic anhydrase II, IV, and XII; β1- and β2 adrenergic receptors; acetylcholinesterase; Na⁺/K⁺-ATPase subunits; and Na—K-2Cl cotransporter. According to the present invention, interfering RNAs provided exogenously or expressed endogenously effect silencing of ocular hypertension target mRNA in ocular tissue(s).

Carbonic anhydrase catalyzes reversible hydration of carbon dioxide and appears to play a role in the regulation of aqueous humor formation. Carbonic anhydrase inhibitors lower pressure in the eye by reducing the amount of fluid produced. Carbonic anhydrase inhibitors are available as eyedrops (dorzolamide, brinzolamide) or tablets/capsules (acetazolamide, methazolamide). The eyedrops are associated with fewer side effects than the tablets or capsules and are better tolerated by many patients. AZOPT® (brinzolamide) ophthalmic suspension 1% is a topical carbonic anhydrase inhibitor (Alcon Laboratories, Inc., Fort Worth, Tex.).

Ophthalmic β-blockers lower pressure in the eye by reducing the amount of fluid produced in the eye. These drugs are divided into two classes: the nonselective beta-blockers (timolol, levobunolol, metipranolol, carteolol) and the β-1 selective blockers (betaxolol). The usual dosage is one drop in each eye once or twice a day, depending on the drug used. An example of this product is BETOPTIC S® (betaxolol HCl) ophthalmic suspension 0.25% (Alcon Laboratories, Inc., Fort Worth, Tex.).

Inhibitors of acetylcholinesterase preserve acetylcholine at the receptor site by blocking the enzyme responsible for its hydrolysis, acetylcholinesterase. Acetylcholine accumulates at the receptor, producing a reduction in intraocular pressure by contraction of the ciliary muscle, similar to the action of direct-acting cholinergic agonists.

Na⁺/K⁺-ATPase inhibitors such as ouabain, nitric oxide donors, and endothelin decrease the activity of Na⁺/K⁺-ATPase, the driving force for aqueous humour formation by the ciliary process.

Chloride transport inhibitors such as ethacrynic acid alter trabecular meshwork cell volume to increase outflow facility.

Nucleic acid sequences cited herein are written in a 5′ to 3′ direction unless indicated otherwise. The term “nucleic acid,” as used herein, refers to either DNA or RNA or a modified form thereof comprising the purine or pyrimidine bases present in DNA (adenine “A,” cytosine “C,” guanine “G,” thymine “T”) or in RNA (adenine “A,” cytosine “C,” guanine “G,” uracil “U”). Interfering RNAs provided herein may comprise “T” bases, particularly at 3′ ends, even though “T” bases do not naturally occur in RNA. “Nucleic acid” includes the terms “oligonucleotide” and “polynucleotide” and can refer to a single-stranded molecule or a double-stranded molecule. A double-stranded molecule is formed by Watson-Crick base pairing between A and T bases, C and G bases, and between A and U bases. The strands of a double-stranded molecule may have partial, substantial or full complementarity to each other and will form a duplex hybrid, the strength of bonding of which is dependent upon the nature and degree of complementarity of the sequence of bases.

An mRNA sequence is readily deduced from the sequence of the corresponding DNA sequence. For example, SEQ ID NO:1 provides the sense strand sequence of DNA corresponding to the mRNA for carbonic anhydrase II. The mRNA sequence is identical to the DNA sense strand sequence with the “T” bases replaced with “U” bases.

Therefore, the mRNA sequence of carbonic anhydrase II is known from SEQ ID NO:1, the mRNA sequence of carbonic anhydrase IV is known from SEQ ID NO:2, the mRNA sequence of β1-adrenergic receptor is known from SEQ ID NO:3, the mRNA sequence of β2-adrenergic receptor is known from SEQ ID NO:4, the mRNA sequence of acetylcholinesterase splice variant E4-E5 is known from SEQ ID NO:5, the mRNA sequence of Na⁺/K⁺-ATPase α2 is known from SEQ ID NO:6, the mRNA sequence of Na—K-2Cl cotransporter A1 is known from SEQ ID NO:7, the mRNA sequence of carbonic anhydrase XII, variant 1 is known from SEQ ID NO:101, the mRNA sequence of acetylcholinesterase splice variant E4-E6 is known from SEQ ID NO:123, the mRNA sequence of Na⁺/K⁺-ATPase al, variant 1, is known from SEQ ID NO:124, the mRNA sequence of Na⁺/K⁺-ATPase α1, variant 2, is known from SEQ ID NO:125, the mRNA sequence of Na⁺/K⁺-ATPase α3 is known from SEQ ID NO:126, the mRNA sequence of Na⁺/K⁺-ATPase α4, variant 1, is known from SEQ ID NO:127, the mRNA sequence of Na⁺/K⁺-ATPase α4, variant 2, is known from SEQ ID NO:128, the mRNA sequence of Na⁺/K⁺-ATPase β1, variant 1, is known from SEQ ID NO:129, the mRNA sequence of Na⁺/K⁺-ATPase β1, variant 2, is known from SEQ ID NO:130, the mRNA sequence of Na⁺/K⁺-ATPase β2, is known from SEQ ID NO:131, the mRNA sequence of Na⁺/K⁺-ATPase β3 is known from SEQ ID NO:132, the mRNA sequence of Na—K-2Cl cotransporter A2 is known from SEQ ID NO:133, and the mRNA sequence of carbonic anhydrase XII, variant 2, is known from SEQ ID NO:134.

Carbonic anhydrases II, IV, and XII mRNA (CA2, CA4, and CA12): Carbonic anhydrases (CAs) II, IV and XII are members of a large family of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide as described by the GenBank database of the National Center for Biotechnology Information at ncbi.nlm.nih.gov. Carbonic anhydrases are involved in crucial physiological processes such as respiration and transport of CO₂/bicarbonate between metabolizing tissues and the lungs, pH and CO₂ homeostasis, electrolyte secretion in a variety of tissues and organs, biosynthetic reactions (such as gluconeogenesis, lipogenesis and ureagenesis), bone resorption, calcification, and tumorigenicity.

Fourteen different carbonic anhydrase isozymes have been identified with different subcellular localizations and tissue distributions. Carbonic anhydrase II is a cytosolic isozyme, whereas carbonic anhydrases IV and XII are membrane-bound. Two transcript variants encoding different isoforms have been identified for the CA-XII gene; variant 1 encodes the longer isoform while variant 2 is lacking one of the internal coding exons compared to transcript variant 1 thereby missing an 11 amino acid segment compared to isoform 1. Systemic carbonic anhydrase inhibitors (CAIs) are useful in reducing the elevated intraocular pressure (IOP) that is characteristic of glaucoma Inhibition of the isozymes present in the ciliary process (the sulfonamide susceptible isozymes CA II and CA IV) reduces the rate of bicarbonate and aqueous humor secretion, which leads to a 25-30% decrease in IOP. However, inhibition of various CA isozymes present in extraocular tissues leads to side effects including numbness and tingling of extremities, metallic taste, depression, fatigue, malaise, weight loss, decreased libido, gastrointestinal irritation, metabolic acidosis, renal calculi, and transient myopia.

The GenBank database provides the DNA sequence for CA2 as accession no. NM_(—)000067, provided in the “Sequence Listing” as SEQ ID NO:1. SEQ ID NO:1 provides the sense strand sequence of DNA that corresponds to the mRNA encoding CAII (with the exception of “T” bases for “U” bases). The coding sequence for CAII is from nucleotides 66-848.

Equivalents of the above cited CA2 mRNA sequence are alternative splice forms, allelic forms, isozymes, or a cognate thereof. A cognate is a CA2 mRNA from another mammalian species that is homologous to SEQ ID NO:1 (i.e., an ortholog). CA2 nucleic acid sequences related to SEQ ID NO:1 include those having GenBank accession numbers M77181, X03251, BC011949, BC035424, CR536526, CR541875, J03037, M36532, S69526, and Y00339.

The GenBank database provides the DNA sequence for CA4 as accession no. NM_(—)000717, provided in the “Sequence Listing” as SEQ ID NO:2. SEQ ID NO:2 provides the sense strand sequence of DNA that corresponds to the mRNA encoding CAIV (with the exception of “T” bases for “U” bases). The coding sequence for CAIV is from nucleotides 47-985.

Equivalents of the above cited CA4 mRNA sequence are alternative splice forms, allelic forms, isozymes, or a cognate thereof. A cognate is a CA4 mRNA from another mammalian species that is homologous to SEQ ID NO:2 (i.e., an ortholog). CA4 nucleic acid sequences related to SEQ ID NO:2 include those having GenBank accession numbers L10955, BC057792, BC069649, BC074768, CR541766, and M83670.

The GenBank database provides the DNA sequence for CA12, variant 1, as accession no. NM_(—)001218, provided in the “Sequence Listing” as SEQ ID NO:101. SEQ ID NO:101 provides the sense strand sequence of DNA that corresponds to the mRNA encoding CAXII, variant 1 (with the exception of “T” bases for “U” bases). The coding sequence for CAXII, variant 1, is from nucleotides 157-1221.

Equivalents of the above cited CA12, variant 1 mRNA sequence are alternative splice forms, allelic forms, isozymes, or a cognate thereof. A cognate is a CA12 mRNA from another mammalian species that is homologous to SEQ ID NO:101 (i.e., an ortholog).

The GenBank database provides the DNA sequence for CA12, variant 2, as accession no. NM_(—)206925, provided in the “Sequence Listing” as SEQ ID NO:134. SEQ ID NO:134 provides the sense strand sequence of DNA that corresponds to the mRNA encoding CAXII, variant 2 (with the exception of “T” bases for “U” bases). The coding sequence for CAXII, variant 2, is from nucleotides 157-1188. Variant 2 lacks an internal coding exon compared to variant 1.

Equivalents of the above cited CA12, variant 2 mRNA sequence are alternative splice forms, allelic forms, isozymes, or a cognate thereof. A cognate is a CA12 mRNA from another mammalian species that is homologous to SEQ ID NO:134 (i.e., an ortholog).

Adrenergic Receptors-β1 and -β2 mRNA (ADRB1 and ADRB2): The adrenergic receptors (subtypes α1, α2, β1, and β2) are a prototypic family of G protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine as described by the GenBank database of the National Center for Biotechnology Information at ncbi.nlm.nih.gov.

The GenBank database provides the DNA sequence for ADRB1 as accession no. NM_(—)000684, provided in the “Sequence Listing” as SEQ ID NO:3. SEQ ID NO:3 provides the sense strand sequence of DNA that corresponds to the mRNA encoding β1-adrenergic receptor (with the exception of “T” bases for “U” bases). The coding sequence for β1-adrenergic receptor is from nucleotides 87-1520.

Equivalents of the above cited ADRB1 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ADRB1 mRNA from another mammalian species that is homologous to SEQ ID NO:3 (i.e., an ortholog). ADRB1 nucleic acid sequences related to SEQ ID NO:3 include those having GenBank accession numbers AF169006, AF169007, AY567837, and J03019.

The GenBank database provides the DNA sequence for ADRB2 as accession no. NM_(—)000024, provided below as SEQ ID NO:4. SEQ ID NO:4 provides the sense strand sequence of DNA that corresponds to the mRNA encoding β2-adrenergic receptor (with the exception of “T” bases for “U” bases). The coding sequence for β2-adrenergic receptor is from nucleotides 220-1461.

Equivalents of the above cited ADRB2 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ADRB2 mRNA from another mammalian species that is homologous to SEQ ID NO:4 (i.e., an ortholog). ADRB2 nucleic acid sequences related to SEQ ID NO:4 include those having GenBank accession numbers AF022953, AF022954, AF022955, AF022956, AF169225, AF202305, AF203386, AY011291, J02960, Y00106, AY136741, BC012481, BC063486, BC073856, M15169, and X04827.

Acetylcholinesterase mRNA splice variants E4-E6 and E4-E5 (ACHE): As described by the GenBank database of the National Center for Biotechnology Information at ncbi.nlm.nih.gov, acetylcholinesterase hydrolyzes the neurotransmitter acetylcholine at neuromuscular junctions and brain cholinergic synapses, and thus terminates signal transmission. It is also found on red blood cell membranes, where it constitutes the Yt blood group antigen. Acetylcholinesterase exists in multiple molecular forms which possess similar catalytic properties, but differ in their oligomeric assembly and mode of cell attachment to the cell surface. It is encoded by the single ACHE gene, and the structural diversity in the gene products arises from alternative mRNA splicing, and post-translational associations of catalytic and structural subunits. The major form of acetylcholinesterase found in brain, muscle and other tissues is the hydrophilic species, which forms disulfide-linked oligomers with collagenous, or lipid-containing structural subunits. The other, alternatively spliced form, expressed primarily in the erythroid tissues, differs at the C-terminal end, and contains a cleavable hydrophobic peptide with a GPI-anchor site. It associates with the membranes through the phosphoinositide (PI) moieties added post-translationally. The splice variant E4-E6 is the major transcript and results from the splicing of exon 4 to exon 6. The splice variant E4-E5 results from alternative splicing of exon 4 to exon 5.

The GenBank database provides the DNA sequence for ACHE splice variant E4-E5 as accession no. NM_(—)015831, provided in the “Sequence Listing” as SEQ ID NO:5. SEQ ID NO:5 provides the sense strand sequence of DNA that corresponds to the mRNA encoding acetylcholinesterase E4-E5 (with the exception of “T” bases for “U” bases). The coding sequence for acetylcholinesterase E4-E5 is from nucleotides 95-1948.

Equivalents of the above cited ACHE mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ACHE mRNA from another mammalian species that is homologous to SEQ ID NO:5 (i.e., an ortholog). ACHE nucleic acid sequences related to SEQ ID NO:5 include those having GenBank accession numbers AC011895, AF002993, AF312032, AY750146, CH236956, L06484, L42812, S71129, AF334270, BC026315, BC036813, M55040 and NM_(—)000665.

The GenBank database provides the DNA sequence for ACHE splice variant E4-E6 as accession no. NM_(—)000665, provided in the “Sequence Listing” as SEQ ID NO:123. SEQ ID NO:123 provides the sense strand sequence of DNA that corresponds to the mRNA encoding acetylcholinesterase E4-E6 variant (with the exception of “T” bases for “U” bases). The coding sequence for acetylcholinesterase E4-E6 is from nucleotides 95-1939.

Equivalents of the above cited ACHE mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ACHE mRNA from another mammalian species that is homologous to SEQ ID NO:123 (i.e., an ortholog). ACHE nucleic acid sequences related to SEQ ID NO:123 include those having GenBank accession numbers NM_(—)015831, AC011895, AF002993, AF312032, AY750146, CH236956, L06484, L42812, S71129, AF334270, BC026315, BC036813, and M55040.

Na⁺/K⁺-ATPase α and β mRNA (ATP1-A1 variant 1, -A1 variant 2, -A2, -A3, -A4 variant 1, -A4 variant 2, -B1 variant 1, -B1 variant 2, -B2, and -B3): As described by the GenBank database, the proteins encoded by these genes belong to the family of P-type cation transport ATPases, and to the subfamily of Na+/K+-ATPases. Na+/K+-ATPase is an integral membrane protein responsible for establishing and maintaining the electrochemical gradients of Na and K ions across the plasma membrane. These gradients are essential for osmoregulation, for sodium-coupled transport of a variety of organic and inorganic molecules, and for electrical excitability of nerve and muscle. This enzyme is composed of two subunits, a large catalytic subunit (a or A) and a smaller glycoprotein subunit (β or B). The catalytic subunit of Na+/K+-ATPase is encoded by multiple genes.

The GenBank database provides the DNA sequence for ATP1A1 variant 1 as accession no. NM_(—)000701, provided in the “Sequence Listing” as SEQ ID NO:124. SEQ ID NO:124 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase subunit A1 variant 1 (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase subunit A1 variant 1 is from nucleotides 299-3370.

Equivalents of the above cited ATP1A1 variant 1 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1A1 variant 1 mRNA from another mammalian species that is homologous to SEQ ID NO:124 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1A1 variant 2 as accession no. NM_(—)001001586, provided in the “Sequence Listing” as SEQ ID NO:125. SEQ ID NO:125 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase subunit A1 variant 2 (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase subunit A1 variant 2 is from nucleotides 299-2344.

Equivalents of the above cited ATP1A1 variant 2 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1A1 variant 2 mRNA from another mammalian species that is homologous to SEQ ID NO:125 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1A2 as accession no. NM_(—)000702, provided in the “Sequence Listing” as SEQ ID NO:6. SEQ ID NO:6 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase A2 subunit (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase A2 subunit is from nucleotides 105-3167.

Equivalents of the above cited ATP1A2 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1A2 mRNA from another mammalian species that is homologous to SEQ ID NO:6 (i.e., an ortholog). ATP1A2 nucleic acid sequences related to SEQ ID NO:6 include those having GenBank accession numbers J05096, M27578, AB018321, AK091617, AK124581, AK126573, AL831991, AL831997, BC013680, BC047533, BC052271, M16795, and Y07494.

The GenBank database provides the DNA sequence for ATP1A3 as accession no. NM_(—)152296, provided in the “Sequence Listing” as SEQ ID NO:126. SEQ ID NO:126 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase A3 subunit (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase A3 subunit is from nucleotides 155-3196.

Equivalents of the above cited ATP1A3 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1A3 mRNA from another mammalian species that is homologous to SEQ ID NO:126 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1A4 variant 1 as accession no. NM_(—)144699, provided in the “Sequence Listing” as SEQ ID NO:127. SEQ ID NO:127 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase A4 subunit variant 1 (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase A4 subunit variant 1 is from nucleotides 469-3558.

Equivalents of the above cited ATP1A4 variant 1 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1A4 variant 1 mRNA from another mammalian species that is homologous to SEQ ID NO:127 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1A4 variant 2 as accession no. NM_(—)001001734, provided in the “Sequence Listing” as SEQ ID NO:128. SEQ ID NO:128 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase A4 subunit variant 2 (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase A4 subunit variant 2 is from nucleotides 111-608.

Equivalents of the above cited ATP1A4 variant 2 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1A4 variant 2 mRNA from another mammalian species that is homologous to SEQ ID NO:128 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1B1 variant 1 as accession no. NM_(—)001677, provided in the “Sequence Listing” as SEQ ID NO:129. SEQ ID NO:129 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase B1 subunit variant 1 (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase B1 subunit variant 1 is from nucleotides 122-1033.

Equivalents of the above cited ATP1B1 variant 1 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1B1 variant 1 mRNA from another mammalian species that is homologous to SEQ ID NO:129 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1B1 variant 2 as accession no. NM_(—)001001787, provided in the “Sequence Listing” as SEQ ID NO:130. SEQ ID NO:130 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase B1 subunit variant 2 (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase B1 subunit variant 2 is from nucleotides 122-1027.

Equivalents of the above cited ATP1B1 variant 2 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1B1 variant 2 mRNA from another mammalian species that is homologous to SEQ ID NO:130 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1B2 as accession no. NM_(—)001678, provided in the “Sequence Listing” as SEQ ID NO:131. SEQ ID NO:131 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase B2 subunit (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase B2 subunit is from nucleotides 584-1456.

Equivalents of the above cited ATP1B2 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1B2 mRNA from another mammalian species that is homologous to SEQ ID NO:131 (i.e., an ortholog).

The GenBank database provides the DNA sequence for ATP1B3 as accession no. NM_(—)001679, provided in the “Sequence Listing” as SEQ ID NO:132. SEQ ID NO:132 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na⁺/K⁺-ATPase B3 subunit (with the exception of “T” bases for “U” bases). The coding sequence for Na⁺/K⁺-ATPase B3 subunit is from nucleotides 175-1014.

Equivalents of the above cited ATP1B3 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is an ATP1B3 mRNA from another mammalian species that is homologous to SEQ ID NO:132 (i.e., an ortholog).

Na—K-2Cl cotransporter mRNA (SLC12A1 and SLC12A2): The sodium-potassium-chloride cotransporter (Na—K-2Cl cotransporter or NKCC) facilitates the coupled cotransport of Na⁺, K⁺, and Cl⁻ ions across the plasma membrane. There are two isoforms: NKCC1 and NKCC2. NKCC1 is expressed in most tissues, including the eye. In contrast, NKCC2 is expressed primarily in the kidney, however, there is evidence for lower level expression of this isoform in the eye as well. NKCC1 is encoded by the SLC12A2 gene (solute carrier family 12, member 2) and NKCC2 is encoded by the SLC12A1 gene. Trabecular meshwork cells possess a robust Na—K-2Cl cotransporter. The activity of this cotransporter is modulated by neurotransmitters and hormones such as norepinephrine, which reduces cotransport activity, or vasopressin, which increases cotransport activity.

The GenBank database provides the DNA sequence for SLC12A1 as accession no. NM_(—)000338, provided in the “Sequence Listing” as SEQ ID NO:7. SEQ ID NO:7 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na—K-2Cl cotransporter NKCC2 (with the exception of “T” bases for “U” bases). The coding sequence for Na—K-2Cl cotransporter NKCC2 is from nucleotides 20-3319.

Equivalents of the above cited Na—K-2Cl NKCC2 cotransporter mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is a Na—K-2Cl cotransporter NKCC2 mRNA from another mammalian species that is homologous to SEQ ID NO:7 (i.e., an ortholog). SLC12A1 nucleic acid sequences related to SEQ ID NO:7 include those having GenBank accession numbers AJ005332, AJ005333, AB032525, AB032527, BC040138, BX647067, BX647484, and U58130.

The GenBank database provides the DNA sequence for SLC12A2 as accession no. NM_(—)001046, provided in the “Sequence Listing” as SEQ ID NO:133. SEQ ID NO:133 provides the sense strand sequence of DNA that corresponds to the mRNA encoding Na—K-2Cl cotransporter NKCC1 (with the exception of “T” bases for “U” bases). The coding sequence for Na—K-2Cl cotransporter NKCC1 is from nucleotides 165-3803.

Equivalents of the above cited Na—K-2Cl cotransporter NKCC1 mRNA sequence are alternative splice forms, allelic forms, or a cognate thereof. A cognate is a Na—K-2Cl cotransporter NKCC1 mRNA from another mammalian species that is homologous to SEQ ID NO:133 (i.e., an ortholog).

Attenuating expression of an mRNA: The phrase, “attenuating expression of an mRNA,” as used herein, means administering or expressing an amount of interfering RNA (e.g., an siRNA) to reduce translation of the target mRNA into protein, either through mRNA cleavage or through direct inhibition of translation. The reduction in expression of the target mRNA or the corresponding protein is commonly referred to as “knock-down” and is reported relative to levels present following administration or expression of a non-targeting control RNA (e.g., a non-targeting control siRNA). Knock-down of expression of an amount including and between 50% and 100% is contemplated by embodiments herein. However, it is not necessary that such knock-down levels be achieved for purposes of the present invention. In one embodiment, a single interfering RNA targeting one of the ocular hypertension targets is administered to lower IOP. In other embodiments, two or more interfering RNAs targeting the same ocular hypertension target (e.g., CA2) are administered to lower IOP. In still other embodiments, two or more interfering RNAs targeting multiple hypertension targets (e.g., CA2 and ADRB2) are administered to lower IOP.

Knock-down is commonly assessed by measuring the mRNA levels using quantitative polymerase chain reaction (qPCR) amplification or by measuring protein levels by western blot or enzyme-linked immunosorbent assay (ELISA). Analyzing the protein level provides an assessment of both mRNA cleavage as well as translation inhibition. Further techniques for measuring knock-down include RNA solution hybridization, nuclease protection, northern hybridization, gene expression monitoring with a microarray, antibody binding, radioimmunoassay, and fluorescence activated cell analysis.

Inhibition of targets cited herein is also inferred in a human or mammal by observing an improvement in a glaucoma symptom such as improvement in intraocular pressure, improvement in visual field loss, or improvement in optic nerve head changes, for example.

Interfering RNA of embodiments of the invention appear to act in a catalytic manner for cleavage of target mRNA, i.e., interfering RNA is able to effect inhibition of target mRNA in substoichiometric amounts. As compared to antisense therapies, significantly less interfering RNA is required to provide a therapeutic effect under such cleavage conditions.

Interfering RNA: In one embodiment of the invention, interfering RNA (e.g., siRNA) has a sense strand and an antisense strand, and the sense and antisense strands comprise a region of at least near-perfect contiguous complementarity of at least 19 nucleotides. In a further embodiment of the invention, the interfering RNA comprises a region of at least 13, 14, 15, 16, 17, or 18 contiguous nucleotides having percentages of sequence complementarity to or, having percentages of sequence identity with, the penultimate 13, 14, 15, 16, 17, or 18 nucleotides, respectively, of the 3′ end of the corresponding target sequence within an mRNA.

The length of each strand of the interfering RNA comprises 19 to 49 nucleotides, and may comprise a length of 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides.

The antisense strand of an siRNA is the active guiding agent of the siRNA in that the antisense strand is incorporated into RISC, thus allowing RISC to identify target mRNAs with at least partial complementarity to the antisense siRNA strand for cleavage or translational repression.

In the present invention, interfering RNA target sequences (e.g., siRNA target sequences) within a target mRNA sequence are selected using available design tools. Interfering RNAs corresponding to these target sequences are then tested by transfection of cells expressing the target mRNA followed by assessment of knockdown as described above. Interfering RNAs that produce a knockdown in expression of between 50% and 100% are selected for further analysis.

Techniques for selecting target sequences for siRNAs are provided by Tuschl, T. et al., “The siRNA User Guide,” revised May 6, 2004, available on the Rockefeller University web site; by Technical Bulletin #506, “siRNA Design Guidelines,” Ambion Inc. at Ambion's web site; and by other web-based design tools at, for example, the Invitrogen, Dharmacon, Integrated DNA Technologies, Genscript, or Proligo web sites. Initial search parameters can include G/C contents between 35% and 55% and siRNA lengths between 19 and 27 nucleotides. The target sequence may be located in the coding region or in the 5′ or 3′ untranslated regions of the mRNA.

An embodiment of a 19-nucleotide DNA target sequence for carbonic anyhdrase II is present at nucleotides 232 to 250 of SEQ ID NO:1:

5′-CCCTGAGGATCCTCAACAA-3′. SEQ ID NO: 8 An siRNA of the invention for targeting a corresponding mRNA sequence of SEQ ID NO:8 and having 21-nucleotide strands and a 2-nucleotide 3′ overhang is:

  5′-CCCUGAGGAUCCUCAACAANN-3′ SEQ ID NO: 9 3′-NNGGGACUCCUAGGAGUUGUU-5′. SEQ ID NO: 10 Each “N” residue can be any nucleotide (A, C, G, U, T) or modified nucleotide. The 3′ end can have a number of “N” residues between and including 1, 2, 3, 4, 5, and 6. The “N” residues on either strand can be the same residue (e.g., UU, AA, CC, GG, or TT) or they can be different (e.g., AC, AG, AU, CA, CG, CU, GA, GC, GU, UA, UC, or UG). The 3′ overhangs can be the same or they can be different. In one embodiment, both strands have a 3′UU overhang.

An siRNA of the invention for targeting a corresponding mRNA sequence of SEQ ID NO:8 and having 21-nucleotide strands and a 3′UU overhang on each strand is:

  5′-CCCUGAGGAUCCUCAACAAUU-3′ SEQ ID NO: 11 3′-UUGGGACUCCUAGGAGUUGUU-5′. SEQ ID NO: 12

The interfering RNA may also have a 5′ overhang of nucleotides or it may have blunt ends. An siRNA of the invention for targeting a corresponding mRNA sequence of SEQ ID NO:8 and having 19-nucleotide strands and blunt ends is:

5′-CCCUGAGGAUCCUCAACAA-3′ SEQ ID NO: 722 3′-GGGACUCCUAGGAGUUGUU-5′. SEQ ID NO: 723

The strands of a double-stranded interfering RNA (e.g., an siRNA) may be connected to form a hairpin or stem-loop structure (e.g., an shRNA). An shRNA of the invention targeting a corresponding mRNA sequence of SEQ ID NO:8 and having a 19 bp double-stranded stem region and a 3′UU overhang is:

N is a nucleotide A, T, C, G, U, or a modified form known by one of ordinary skill in the art. The number of nucleotides N in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11, or the number of nucleotides N is 9. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop. Examples of oligonucleotide sequences that can be used to form the loop include 5′-UUCAAGAGA-3′ (Brummelkamp, T. R. et al. (2002) Science 296: 550) and 5′-UUUGUGUAG-3′ (Castanotto, D. et al. (2002) RNA 8:1454). It will be recognized by one of skill in the art that the resulting single chain oligonucleotide forms a stem-loop or hairpin structure comprising a double-stranded region capable of interacting with the RNAi machinery.

The siRNA target sequence identified above can be extended at the 3′ end to facilitate the design of dicer-substrate 27-mer duplexes. Extension of the 19-nucleotide DNA target sequence (SEQ ID NO:8) identified in the carbonic anhydrase II DNA sequence (SEQ ID NO:1) by 6 nucleotides yields a 25-nucleotide DNA target sequence present at nucleotides 232 to 256 of SEQ ID NO:1:

5′-CCCTGAGGATCCTCAACAATGGTCA-3′. SEQ ID NO: 724 A dicer-substrate 27-mer duplex of the invention for targeting a corresponding mRNA sequence of SEQ ID NO:724 is:

  5′-CCCUGAGGAUCCUCAACAAUGGUCA-3′ SEQ ID NO: 718 3′-UUGGGACUCCUAGGAGUUGUUACCAGU-5′. SEQ ID NO: 719 The two nucleotides at the 3′ end of the sense strand (i.e., the CA nucleotides of SEQ ID NO:718) may be deoxynucleotides for enhanced processing. Design of dicer-substrate 27-mer duplexes from 19-21 nucleotide target sequences, such as provided herein, is further discussed by the Integrated DNA Technologies (IDT) website and by Kim, D.-H. et al., (February, 2005) Nature Biotechnology 23:2; 222-226.

When interfering RNAs are produced by chemical synthesis, phosphorylation at the 5′ position of the nucleotide at the 5′ end of one or both strands (when present) can enhance siRNA efficacy and specificity of the bound RISC complex but is not required since phosphorylation can occur intracellularly.

Table 1 lists examples of siRNA target sequences within the CA2, CA4, and CA12 variant 1 and variant 2 DNA sequences (SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:101, and SEQ ID NO:134, respectively) from which siRNAs of the present invention are designed in a manner as set forth above. CA2, CA4, and CA12 variant 1 and variant 2 encode carbonic anhydrase II, IV, and XII variant 1 and 2, respectively.

TABLE 1 CA2, CA4, and CA12 Target Sequences for siRNAs # of Starting Nucleotide with reference to CA2 Target Sequence SEQ ID NO: 1 SEQ ID NO: CCCTGAGGATCCTCAACAA 232 8 GGGCCTTCAGAAAGTTGTT 527 14 GCGAGCAGGTGTTGAAATT 721 15 GGTGTTGAAATTCCGTAAA 728 16 GCCACTGAAGAACAGGCAA 809 17 CCACTGAAGAACAGGCAAA 810 18 CCCATAGTCTGTATCCAAA 855 19 CCATAGTCTGTATCCAAAT 856 20 GGTGATTTGGACCCTGGTT 921 21 GGGTGATGAGCACTCACAA 1139 22 GAAGGTTGGCAGCGCTAAA 506 83 ATGTGCTGGATTCCATTAA 547 84 TGTGCTGGATTCCATTAAA 548 85 CCGTAAACTTAACTTCAAT 740 86 GATCTACCTTGGTGATTTG 911 87 GACCAATTGTCATGCTTGA 1009 88 GGTGATGAGCACTCACAAT 1140 89 CACTCACAATTGTTGACTA 1149 90 ACTCACAATTGTTGACTAA 1150 91 CTCACAATTGTTGACTAAA 1151 92 AGGAAAGTAGAATGGTTGA 1188 93 GTAGAATGGTTGAGTGCAA 1194 94 TAGAATGGTTGAGTGCAAA 1195 95 CAAGATAAATTGAGCTAGT 1223 96 AGTTAAGGCAAATCAGGTA 1239 97 GAGTTGTGATACAGAGTAT 1456 98 AGTTGTGATACAGAGTATA 1457 99 GTTGTGATACAGAGTATAT 1458 100 GACCTGAGCACTGGCATAA 100 135 TGACATCGACACTCATACA 158 136 ACACTCATACAGCCAAGTA 166 137 ACAATGGTCATGCTTTCAA 247 138 AGGACAAAGCAGTGCTCAA 286 139 GATGGCACTTACAGATTGA 318 140 GCACTTACAGATTGATTCA 322 141 ACAGATTGATTCAGTTTCA 328 142 ACAAGGTTCAGAGCATACT 371 143 CAGAACTTCACTTGGTTCA 412 144 ACTGGCCGTTCTAGGTATT 482 145 TTGAAGGTTGGCAGCGCTA 504 146 TGAAGGTTGGCAGCGCTAA 505 147 TTGTTGATGTGCTGGATTC 541 148 GAAATTCCGTAAACTTAAC 734 149 CCGAAGAACTGATGGTGGA 772 150 GAACTGATGGTGGACAACT 777 151 TGAAGAACAGGCAAATCAA 814 152 CTTACTTGATAGACTTACT 972 153 TGTGAAGACTAGACCAATT 998 154 TTGAGCTAGTTAAGGCAAA 1232 155 GGATGGCACTTACAGATTG 317 720 GAAATATGCTGCAGAACTT 401 721 # of Starting  Nucleotide with reference to CA4 Target Sequence SEQ ID NO: 2 SEQ ID NO: TCGTCACCACCAAGGCAAA 213 23 GCTTCTTCTTCTCTGGCTA 252 24 TCTTCTCTGGCTACGATAA 258 25 GGCTACGATAAGAAGCAAA 266 26 GGTCCGACTTGCCATATAA 399 27 GGAGATGCACATAGTACAT 457 28 GCACATAGTACATGAGAAA 463 29 GACATCGAGGAATGTGAAA 490 30 GGTGGAGGCACTGTCTAAT 595 31 GGGACTTTAGGCATGATTA 1064 32 ACACTGGTGCTACGAGGTT 109 156 CTGGTGCTACGAGGTTCAA 112 157 GTTCAAGCCGAGTCCTCCA 125 158 TTCAAGCCGAGTCCTCCAA 126 159 CCTGCTTGGTGCCAGTCAA 150 160 TCTCTGGCTACGATAAGAA 261 161 TGGCTACGATAAGAAGCAA 265 162 GCAAACGTGGACTGTCCAA 280 163 TGGTCCGACTTGCCATATA 398 164 CCATGGAGATGCACATAGT 453 165 AGATGCACATAGTACATGA 459 166 TGCACATAGTACATGAGAA 462 167 ATAGTACATGAGAAAGAGA 467 168 CATCGAGGAATGTGAAAGA 492 169 TTGCGGTGCTGGCCTTTCT 534 170 GAACAGATCCTGGCATTCT 785 171 TCTCTCAGAAGCTGTACTA 801 172 AGGAACAGACAGTGAGCAT 825 173 GAACAGACAGTGAGCATGA 827 174 GGCAGCGCACGGTGATAAA 876 175 CAGCCTCTCTGTTGCCTCA 1003 176 TGTTGCCTCAGCTCTCCAA 1012 177 CA12, variant # of Starting  1 and 2 Nucleotide with Common Target reference to Sequences SEQ ID NO: 101 SEQ ID NO: TCCTGCTGGTGATCTTAAA 191 102 ACGGTTCCAAGTGGACTTA 239 103 GAGAATAGCTGGTCCAAGA 274 104 AGAATAGCTGGTCCAAGAA 275 105 GTGACATCCTCCAGTATGA 341 106 GCTACAATCTGTCTGCCAA 389 107 CAGTTTCTCCTGACCAACA 412 108 AGTTTCTCCTGACCAACAA 413 109 GACCAACAATGGCCATTCA 423 110 CTCCTTCAATCCGTCCTAT 687 111 CCTTCAATCCGTCCTATGA 689 112 ATCCGTCCTATGACAAGAT 695 113 AGATCTTCAGTCACCTTCA 710 114 CGGAGAGGACCGCTGAATA 791 115 GGAGAGGACCGCTGAATAT 792 116 AGAGGACCGCTGAATATTA 794 117 AGGTCCAGAAGTTCGATGA 983 118 GTTCGATGAGAGGCTGGTA 993 119 TTCGATGAGAGGCTGGTAT 994 120 TCGATGAGAGGCTGGTATA 995 121 TTCAATCCGTCCTATGACA 691 178 # of Starting CA12, variant Nucleotide with 1 Target reference to Sequence SEQ ID NO: 101 SEQ ID NO: TGTACTGCGGCAGGACTGA 1039 122 AGAGCGTGCTTTCAAGTGT 1568 179 GATGTCAAATCGTGGTTTA 2326 180 AAATCGTGGTTTAGATCAA 2332 181 ATGGAATGCTACTAAGATA 2425 182 CTACTAAGATACTCCATAT 2433 183 ACAACGATGGCAAGCCTTA 2844 184 CAACGATGGCAAGCCTTAT 2845 185 TTGCTAGGCAAAGTTACAA 2880 186 TAGGCAAAGTTACAAGTGA 2884 187 AGTTACAAGTGACCTAATG 2891 188 TGTGCACTCAAGACCTCTA 2954 189 GTGCACTCAAGACCTCTAA 2955 190 TGCACTCAAGACCTCTAAC 2956 191 GCACTCAAGACCTCTAACA 2957 192 AGACCTCTAACAGCCTCGA 2964 193 GACCTCTAACAGCCTCGAA 2965 194 TGCCATTAGCATGCCTCAT 3006 195 GCCATTAGCATGCCTCATG 3007 196 TAGCATGCCTCATGCATCA 3012 197 CATCATCAGATGACAAGGA 3026 198 # of Starting CA12, variant Nucleotide with 2 Target reference to Sequence SEQ ID NO: 134 SEQ ID NO: CTCCTTCAATCCGTCCTAT 687 199 AGAGCGTGCTTTCAAGTGT 1535 200 GATGTCAAATCGTGGTTTA 2293 201 AAATCGTGGTTTAGATCAA 2299 202 ATGGAATGCTACTAAGATA 2392 203 CTACTAAGATACTCCATAT 2400 204 ACAACGATGGCAAGCCTTA 2811 205 CAACGATGGCAAGCCTTAT 2812 206 TTGCTAGGCAAAGTTACAA 2847 207 TAGGCAAAGTTACAAGTGA 2851 208 AGTTACAAGTGACCTAATG 2858 209 TGTGCACTCAAGACCTCTA 2921 210 GTGCACTCAAGACCTCTAA 2922 211 TGCACTCAAGACCTCTAAC 2923 212 GCACTCAAGACCTCTAACA 2924 213 AGACCTCTAACAGCCTCGA 2931 214 GACCTCTAACAGCCTCGAA 2932 215 TGCCATTAGCATGCCTCAT 2973 216 GCCATTAGCATGCCTCATG 2974 217 TAGCATGCCTCATGCATCA 2979 218 CATCATCAGATGACAAGGA 2993 219

Table 2 lists examples of siRNA target sequences within the ADRB1 and ADRB2 DNA sequences (SEQ ID NO:3 and SEQ ID NO:4, respectively) from which siRNAs of the present invention are designed in a manner as set forth above. As noted above, ADRB1 and ADRB2 encode the β1- and β2-adrenergic receptors, respectively.

TABLE 2 ADRB1 and ADRB2 Target Sequences for siRNAs # of Starting Nucleotide with ADRB1 Target reference to Sequence SEQ ID NO: 3 SEQ ID NO: TCCTTCTTCTGCGAGCTGT 468 33 TCGAGACCCTGTGTGTCAT 523 34 GCATCATGGCCTTCGTGTA 799 35 GAACGAGGAGATCTGTGTT 1563 36 ACGAGGAGATCTGTGTTTA 1565 37 GGAGATCTGTGTTTACTTA 1569 38 GATAGCAGGTGAACTCGAA 1593 39 CCCACAATCCTCGTCTGAA 1613 40 CCACAATCCTCGTCTGAAT 1614 41 TCTGAATCATCCGAGGCAA 1626 42 GCAATGTGCTGGTGATCGT 310 220 TGATCGTGGCCATCGCCAA 322 221 AAGTGCTGCGACTTCGTCA 726 222 CGTCCGTAGTCTCCTTCTA 769 223 CCGTAGTCTCCTTCTACGT 772 224 ATCATGGCCTTCGTGTACC 801 225 TCATGGCCTTCGTGTACCT 802 226 CCTCGGAATCCAAGGTGTA 1501 227 TGTGTTTACTTAAGACCGA 1576 228 GTGTTTACTTAAGACCGAT 1577 229 GTTTACTTAAGACCGATAG 1579 230 TTTACTTAAGACCGATAGC 1580 231 TTACTTAAGACCGATAGCA 1581 232 TAAGACCGATAGCAGGTGA 1586 233 ACCGATAGCAGGTGAACTC 1590 234 CGATAGCAGGTGAACTCGA 1592 235 ATAGCAGGTGAACTCGAAG 1594 236 CACAATCCTCGTCTGAATC 1615 237 ACAATCCTCGTCTGAATCA 1616 238 TCATCCGAGGCAAAGAGAA 1632 239 CATCCGAGGCAAAGAGAAA 1633 240 CCACGGACCGTTGCACAAA 1654 241 # of Starting Nucleotide with ADRB2 Target reference to Sequence SEQ ID NO: 4 SEQ ID NO: GCATCGTCATGTCTCTCAT 329 43 GCTGGTCATCACAGCCATT 375 44 CCCTCAAGACGTTAGGCAT 1031 45 GCATCATCATGGGCACTTT 1046 46 CCTAAATTGGATAGGCTAT 1149 47 GCTATGTCAATTCTGGTTT 1163 48 GGAAGACTTTGTGGGCCAT 1371 49 GCCTAGCGATAACATTGAT 1401 50 GGGAGGAATTGTAGTACAA 1426 51 GCTGTGAACATGGACTCTT 1880 52 CACGACGTCACGCAGCAAA 283 242 GATCGCTACTTTGCCATTA 607 243 ATCGCTACTTTGCCATTAC 608 244 TCGCTACTTTGCCATTACT 609 245 GCCATTACTTCACCTTTCA 619 246 TTACTTCACCTTTCAAGTA 623 247 CCATTCAGATGCACTGGTA 722 248 TGATCATGGTCTTCGTCTA 857 249 AGACGTTAGGCATCATCAT 1037 250 TCGTTAACATTGTGCATGT 1091 251 AGGATAACCTCATCCGTAA 1115 252 TCATCCGTAAGGAAGTTTA 1124 253 AAGTTTACATCCTCCTAAA 1136 254 AGTTTACATCCTCCTAAAT 1137 255 TAAATTGGATAGGCTATGT 1151 256 CTATGTCAATTCTGGTTTC 1164 257 GGTACTGTGCCTAGCGATA 1393 258 GTACTGTGCCTAGCGATAA 1394 259 TACTGTGCCTAGCGATAAC 1395 260 GCGATAACATTGATTCACA 1406 261 CGATAACATTGATTCACAA 1407 262 GGAGGAATTGTAGTACAAA 1427 263 GAGGAATTGTAGTACAAAT 1428 264 AGGAATTGTAGTACAAATG 1429 265 CAAATGACTCACTGCTGTA 1442 266 GACCTGAGTCTGCTATATT 1725 267 ACCTGAGTCTGCTATATTT 1726 268 CCATGTATCTACCTCACTA 1756 269 CATGTATCTACCTCACTAT 1757 270 ATGTATCTACCTCACTATT 1758 271 CCTCACTATTCAAGTATTA 1767 272 TAATATATTGCTGCTGGTA 1790 273 AATATATTGCTGCTGGTAA 1791 274 ATATATTGCTGCTGGTAAT 1792 275 TATATTGCTGCTGGTAATT 1793 276 CTGGTAATTTGTATCTGAA 1803 277 GAGTATCTCGGACCTTTCA 1861 278 CGGACCTTTCAGCTGTGAA 1869 279 CGAGCAAAGGTCTAAAGTT 1971 280 GAGCAAAGGTCTAAAGTTT 1972 281 GGTCTAAAGTTTACAGTAA 1979 282

Table 3 lists examples of siRNA target sequences within the ACHE DNA sequences for splice variants E4-E5 and E4-E6 (SEQ ID NO:5 and SEQ ID NO:123, respectively) from which siRNAs of the present invention are designed in a manner as set forth above. As noted above, ACHE encodes acetylcholinesterase.

TABLE 3 ACHE Target Sequences for siRNAs # of Starting Nucleotide with ACHE E4-E5 reference to Target Sequence SEQ ID NO: 5 SEQ ID NO: CCAGAGTGTCTGCTACCAA 382 53 GCTACCAATATGTGGACAC 393 54 CCAATATGTGGACACCCTA 397 55 GCTGGTGTCCATGAACTAC 622 56 TCATCAACGCGGGAGACTT 1131 57 GGTCTACGCCTACGTCTTT 1459 58 GCTACGAGATCGAGTTCAT 1530 59 GCTATAACGGTCAACCATT 2251 60 GGCTGCAAATAAACTGTTA 2885 61 GCTGCAAATAAACTGTTAC 2886 62 AGTGTCTGCTACCAATATG 386 283 AGACAACGAGTCTCTCATC 1231 284 GGCTGTGGTCCTGCATTAC 1315 285 CTTCCTCCTCAAACCGAGA 2047 286 TCCTCCTCAAACCGAGAGA 2049 287 CCTCAAACCGAGAGACTCA 2053 288 TCAAACCGAGAGACTCACA 2055 289 AAACCGAGAGACTCACACT 2057 290 CCACGCCTTTGTTGTTTGA 2125 291 CACGCCTTTGTTGTTTGAA 2126 292 ACGCCTTTGTTGTTTGAAT 2127 293 GGCTATAACGGTCAACCAT 2250 294 TATAACGGTCAACCATTTC 2253 295 CGGTCAACCATTTCTGTCT 2258 296 GTCAACCATTTCTGTCTCT 2260 297 CCGTCTTCCGGTCATTCTT 2318 298 CCTCTCGTCTTTCGCACAT 2395 299 TCTCGTCTTTCGCACATTC 2397 300 TTTCGCACATTCTCCTGAT 2404 301 TTCGCACATTCTCCTGATC 2405 302 AGAACCAGTTCGACCACTA 2643 303 AACCAGTTCGACCACTACA 2645 304 CTGCAAATAAACTGTTACA 2887 305 ACHE E4-E5 # of Starting and E4-E6 Nucleotide with Target Sequences reference to in Common SEQ ID NO: 5 SEQ ID NO: TAGACGCTACAACCTTCCA 366 306 CGCTACAACCTTCCAGAGT 370 307 AGAGTGTCTGCTACCAATA 384 308 GAGTGTCTGCTACCAATAT 385 309 CTGTCCTCGTCTGGATCTA 525 310 ATGGCCGCTTCTTGGTACA 588 311 CGACATCAGTGACGCTGTT 768 312 GCACGTGCTGCCTCAAGAA 1045 313 CACGTGCTGCCTCAAGAAA 1046 314 GAAAGCGTCTTCCGGTTCT 1061 315 TGTGGTAGATGGAGACTTC 1090 316 GACAACGAGTCTCTCATCA 1232 317 AGGCTGTGGTCCTGCATTA 1314 318 GCTGTGGTCCTGCATTACA 1316 319 GTCTACGCCTACGTCTTTG 1460 320 TCTACGCCTACGTCTTTGA 1461 321 CTACGCCTACGTCTTTGAA 1462 322 CGGCTACGAGATCGAGTTC 1528 323 CAGCGACTGATGCGATACT 1607 324 GGCTCAGCAGTACGTTAGT 1705 325 AGTACGTTAGTCTGGACCT 1713 326 # of Starting Nucleotide with ACHE E4-E6 reference to Target Sequence SEQ ID NO: 123 SEQ ID NO: ACATGGTGCACTGGAAGAA 1875 327 AGAACCAGTTCGACCACTA 1890 328 GAACCAGTTCGACCACTAC 1891 329 GGCTATAACACAGACGAGC 2011 330 GCTATAACACAGACGAGCC 2012 331 GCTGCAAATAAACTGTTAC 2133 332 CTGCAAATAAACTGTTACA 2134 333

Table 4 lists examples of siRNA target sequences within the Na⁺/K⁺-ATPase A and B subunit DNA sequences (ATP1A1 variant 1, SEQ ID NO:124; ATP1A1 variant 2, SEQ ID NO:125; ATP1A2, SEQ ID NO:6; ATP1A3, SEQ ID NO:126; ATP1A4 variant 1, SEQ ID NO:127; ATP1A4 variant 2, SEQ ID NO:128; ATP1B1 variant 1, SEQ ID NO:129; ATP1B1 variant 2, SEQ ID NO:130; ATP1B2, SEQ ID NO:131; and ATP1B3, SEQ ID NO:132) from which siRNAs of the present invention are designed in a manner as set forth above.

TABLE 4 ATP1A and ATP1B Target Sequences for siRNAs # of Starting  Nucleotide with ATP1A1 variant 1 reference to Target Sequence SEQ ID NO: 124 SEQ ID NO: GCAATGAGACCGTGGAAGA 2208 334 TGCCAAGGCCTGCGTAGTA 2275 335 TAAAGGACATGACCTCCGA 2307 336 AGCAAGCTGCTGACATGAT 2526 337 ACATGATTCTTCTGGATGA 2538 338 GTCGTCTGATCTTTGATAA 2592 339 CTTATACCTTAACCAGTAA 2628 340 GGATCAACGATGTGGAAGA 2979 341 ACGATGTGGAAGACAGCTA 2985 342 CCGACTTGGTCATCTGTAA 3093 343 TAGGAAAGCACCGCAGCAT 3474 344 AGACGTCCTGGAATGAAGC 3504 345 GACGTCCTGGAATGAAGCA 3505 346 ACGTCCTGGAATGAAGCAT 3506 347 GAAGCATGTAGCTCTATGG 3518 348 ATP1A1 variant # of Starting 1 and Nucleotide with variant 2 Common reference to Target Sequences SEQ ID NO: 124 SEQ ID NO: TTCAGAACAAGGTGATAAA 343 349 TGATGAACTTCATCGTAAA 442 350 GGTGCTATCAGCCGTTGTA 700 351 TCAGCCGTTGTAATCATAA 707 352 GATTCGAAATGGTGAGAAA 811 353 CAGAATCATATCTGCAAAT 907 354 CACGTGGTATTGTTGTCTA 1059 355 CTGCTTAGTGAAGAACTTA 1363 356 GTTTCAGGCTAACCAGGAA 1594 357 CACTCTTAAAGTGCATAGA 1662 358 AGTACCAGTTGTCTATTCA 1758 359 TACCAGTTGTCTATTCATA 1760 360 AGCTGAAAGACGCCTTTCA 1896 361 TCGATAATCTGTGCTTTGT 2037 362 ACAGGAGACCATCCAATCA 2147 363 #of Starting Nucleotide with ATP1A1 variant 2 reference to Target Sequence SEQ ID NO: 125 SEQ ID NO: TAGCCTTGATGAACTTCAT 436 364 TTGATGAACTTCATCGTAA 441 365 GATGAACTTCATCGTAAAT 443 366 CTACTCCTGAATGGATCAA 552 367 GGAGCGATTCTTTGTTTCT 617 368 GTGCTATCAGCCGTTGTAA 701 369 TGCTATCAGCCGTTGTAAT 702 370 GAGCATAAATGCGGAGGAA 832 371 GAAGGCAATGGACCTATGA 2204 372 CCGACTTGGTCATCTGTAA 2291 373 TATATGACGAAGTCAGAAA 2495 374 # of Starting Nucleotide with ATP1A2 Target reference to Sequence SEQ ID NO: 6 SEQ ID NO: CCATCCAACGACAATCTAT 471 63 GCATCATATCAGAGGGTAA 1990 64 CCTCCTCATCTTCATCTAT 3080 65 GGAAGTGAGGTAGTGCCAA 3797 66 GGATGTCACTCATGTACTT 4037 67 GCTCCATGCTGTTCTGAAA 4093 68 GCTGGCCATTGGCTAGAAT 4225 69 GGTCAGAACCTTTGGACAA 4323 70 GCTAGAGGTGGCATGTTTA 5213 71 GCGAGTGCATGGGCTAATT 5285 72 TGGCAATGGATGACCACAA 214 375 TGAACCATCCAACGACAAT 467 376 ACCATCCAACGACAATCTA 470 377 CATCCAACGACAATCTATA 472 378 ATCCAACGACAATCTATAT 473 379 GCAGATCAACGCAGAGGAA 632 380 TGTTTCTTCTCCACCAACT 825 381 CCATAGCAATGGAGATTGA 946 382 AGATGCAAGATGCCTTTCA 1693 383 CTGAATCTGCCATCTGGAA 1767 384 TGAATCTGCCATCTGGAAA 1768 385 ATCGTCTTTGCTCGAACGT 2157 386 CTGCATTGAAGAAGGCTGA 2263 387 ATGAAGCGGCAGCCACGAA 2589 388 TGAAGCGGCAGCCACGAAA 2590 389 GGATGACCGGACCATGAAT 2765 390 GCTGCCTTTCTCTCTTACT 2988 391 TCTATGATGAGGTCCGAAA 3094 392 GTGGAGAAGGAGACATACT 3144 393 TGGAGAAGGAGACATACTA 3145 394 TAGACCTAACTGTGAACAA 3344 395 AGACCTAACTGTGAACAAT 3345 396 TCCACTATGTTGTCTATTT 3418 397 TGAGTGCAAGAGCCTGAGA 3666 398 TGACATGAGTCTCCAGATA 3828 399 GTCGTGGACTCCAGCTCTA 3850 400 TGTCACTCATGTACTTAAT 4040 401 GTCACTCATGTACTTAATA 4041 402 CACTTCACCTTCTGTAATA 4061 403 GTAGAGAGAGACCTAGATA 4882 404 CTAGATAGGTCATGCAAGT 4894 405 AGGTCATGCAAGTGAGAAA 4900 406 TATCAGAAGCAAGGAAGTA 5040 407 TCCGATTAATTGGAGATTA 5114 408 CCGATTAATTGGAGATTAC 5115 409 GATTACTAACTGTGGACAA 5128 410 ATTACTAACTGTGGACAAA 5129 411 TCAGGCACTTTAGAAATAT 5253 412 GGCTAATTATCATCAATCT 5296 413 AGTTTGAGGTACTACCTAT 5375 414 TACTACCTATGTACTTGAA 5384 415 ACTACCTATGTACTTGAAA 5385 416 # of Starting Nucleotide with ATP1A3 Target reference to Sequence SEQ ID NO: 126 SEQ ID NO: TGGCTATGACAGAGCACAA 240 417 GAGGTCTGCCGGAAATACA 272 418 CTCACGCCACCGCCTACCA 362 419 TCGACTGTGATGACGTGAA 1836 420 TGAACTTCACCACGGACAA 1851 421 CCAAGGCCTGCGTGATCCA 2103 422 GGACTTCACCTCCGAGCAA 2137 423 GACTTCACCTCCGAGCAAA 2138 424 ACTTCACCTCCGAGCAAAT 2139 425 TCGACGAGATCCTGCAGAA 2157 426 CGACGAGATCCTGCAGAAT 2158 427 ACGAGATCCTGCAGAATCA 2160 428 GATCTTCGACAACCTAAAG 2425 429 CCATCTCACTGGCGTACGA 2580 430 CTGCCGAAAGCGACATCAT 2601 431 CGGACAAATTGGTCAATGA 2646 432 CAAATTGGTCAATGAGAGA 2650 433 GGATGACCGCACCGTCAAT 2794 434 CACCGTCAATGACCTGGAA 2803 435 ATCTTCGTCTACGACGAAA 3116 436 CTACGACGAAATCCGCAAA 3124 437 ACGACGAAATCCGCAAACT 3126 438 ACGAAATCCGCAAACTCAT 3129 439 CCAAACCTCTCTCCTCTCT 3377 440 # of Starting Nucleotide with ATP1A4 variant 1 reference to Target Sequence SEQ ID NO: 127 SEQ ID NO: GGCACCTGGTTACGCTTCA 113 441 CATGGATGATCACAAATTA 612 442 AATCCTGACTCGAGATGGA 702 443 CCTACAGCATCCAGATATA 833 444 CCGGCTTATCTCTGCACAA 1101 445 AGCTCTGATACCTGGTTTA 1732 446 GCTCTGATACCTGGTTTAT 1733 447 AGGTGATGCTTCCGAGTCA 1836 448 GTACTCAATGAACGATGAA 2070 449 TACTCAATGAACGATGAAA 2071 450 GTGCTAGGCTTCTGCTTCT 2143 451 CATGGTAACAGGAGATCAT 2328 452 TGTGGTGCATGGTGCAGAA 2475 453 TGTTCATCATCCTCGGTAT 2861 454 GTTCATCATCCTCGGTATA 2862 455 GGCTTATGAGTCAGCTGAA 2952 456 GGACCTATGAGCAACGAAA 3203 457 CGGATCTCATCATCTCCAA 3281 458 TGGCTGCATTTCTGTCCTA 3377 459 GCTGCATTTCTGTCCTACA 3379 460 GTATTCTCATCTTCGTCTA 3470 461 TATTCTCATCTTCGTCTAT 3471 462 ACTAAACTCAGCAGATGAA 3554 463 GGCCAGAGATTATAAGTTT 3614 464 GCCAGAGATTATAAGTTTG 3615 465 CCAGAGATTATAAGTTTGA 3616 466 CAGAGATTATAAGTTTGAC 3617 467 ATAAGTTTGACACAACATC 3625 468 TAAGTTTGACACAACATCT 3626 469 TCTGAGACACTAGGATGAA 3642 470 AGACACTAGGATGAATTAT 3646 471 GACACTAGGATGAATTATC 3647 472 AGGATGAATTATCTTGGAT 3653 473 GATGAATTATCTTGGATGA 3655 474 CGTAGCCAGTCTAGACAGT 3797 475 GCCAGTCTAGACAGTAAAT 3801 476 CAGTCTAGACAGTAAATGT 3803 477 AGACAGTAAATGTCTGGAA 3809 478 GACAGTAAATGTCTGGAAA 3810 479 # of Starting Nucleotide with ATP1A4 variant 2 reference to Target Sequence SEQ ID NO: 128 SEQ ID NO: GCTGGATTCTTTACCTACT 126 480 GTGGACCTATGAGCAACGA 251 481 TGGACCTATGAGCAACGAA 252 482 GGACCTATGAGCAACGAAA 253 483 CGGATCTCATCATCTCCAA 331 484 TGGCTGCATTTCTGTCCTA 427 485 GCTGCATTTCTGTCCTACA 429 486 GTATTCTCATCTTCGTCTA 520 487 TATTCTCATCTTCGTCTAT 521 488 CTTCGTCTATGATGAAATC 530 489 ACTACTAAACTCAGCAGAT 601 490 CTACTAAACTCAGCAGATG 602 491 TACTAAACTCAGCAGATGA 603 492 ACTAAACTCAGCAGATGAA 604 493 GGCCAGAGATTATAAGTTT 664 494 GCCAGAGATTATAAGTTTG 665 495 CCAGAGATTATAAGTTTGA 666 496 CAGAGATTATAAGTTTGAC 667 497 ATAAGTTTGACACAACATC 675 498 TAAGTTTGACACAACATCT 676 499 TCTGAGACACTAGGATGAA 692 500 AGACACTAGGATGAATTAT 696 501 GACACTAGGATGAATTATC 697 502 TAGGATGAATTATCTTGGA 702 503 AGGATGAATTATCTTGGAT 703 504 GATGAATTATCTTGGATGA 705 505 TGAATTATCTTGGATGAGA 707 506 CGTAGCCAGTCTAGACAGT 847 507 GCCAGTCTAGACAGTAAAT 851 508 CAGTCTAGACAGTAAATGT 853 509 AGACAGTAAATGTCTGGAA 859 510 GACAGTAAATGTCTGGAAA 860 511 #of Starting  Nucleotide with ATP1B1 variant 1 reference to Target Sequence SEQ ID NO: 129 SEQ ID NO: ACCTACTAGTCTTGAACAA 1096 512 TACTAGTCTTGAACAAACT 1099 513 GGACCTACACTTAATCTAT 1130 514 GACCTACACTTAATCTATA 1131 515 CTGCATTTAATAGGTTAGA 1167 516 CGTAACTGACTTGTAGTAA 1299 517 AGCAAGGTTTGCTGTCCAA 1441 518 TGCTGTCCAAGGTGTAAAT 1450 519 GCTGTCCAAGGTGTAAATA 1451 520 CTGTCCAAGGTGTAAATAT 1452 521 TTAACATACTCCATAGTCT 1564 522 GCCTTGTCCTCCGGTATGT 1746 523 TGTCCTCCGGTATGTTCTA 1750 524 GTCCTCCGGTATGTTCTAA 1751 525 TCCTCCGGTATGTTCTAAA 1752 526 CCATCACTTTGGCTAGTGA 1795 527 ATP1B1 variant #of Starting 1 and Nucleotide with variant 2 Common reference to Target Sequences SEQ ID NO: 129 SEQ ID NO: ACCGGTGGCAGTTGGTTTA 203 528 CCGGTGGCAGTTGGTTTAA 204 529 TTGGTTTAAGATCCTTCTA 214 530 AGATCCTTCTATTCTACGT 222 531 ATCCTTCTATTCTACGTAA 224 532 TCCTTCTATTCTACGTAAT 225 533 CCTTCTATTCTACGTAATA 226 534 GAAATTTCCTTTCGTCCTA 380 535 AACGAGGAGACTTTAATCA 525 536 GAAATTGCTCTGGATTAAA 591 537 ATGAAACTTATGGCTACAA 612 538 TGAAACTTATGGCTACAAA 613 539 AAACTTATGGCTACAAAGA 615 540 GGCAAACCGTGCATTATTA 635 541 GCAAACCGTGCATTATTAT 636 542 ACCGAGTTCTAGGCTTCAA 663 543 CCGAGTTCTAGGCTTCAAA 664 544 TTCTAGGCTTCAAACCTAA 669 545 ATGAGTCCTTGGAGACTTA 699 546 GCAAGCGAGATGAAGATAA 765 547 AGTTGGAAATGTGGAGTAT 790 548 CTGCAGTATTATCCGTACT 839 549 TGCAGTATTATCCGTACTA 840 550 GCAGTATTATCCGTACTAT 841 551 CCGTACAGTTCACCAATCT 900 552 TCACCAATCTTACCATGGA 909 553 AAATTCGCATAGAGTGTAA 933 554 TGTAAGGCGTACGGTGAGA 947 555 # of Starting  Nuleotide with ATP1B1 variant reference to 2 Target Sequence SEQ ID NO: 130 SEQ ID NO: TGTGTTATGCTTGTATTGA 1063 556 GCCTTGTCCTCCGGTATGT 1102 557 TGTCCTCCGGTATGTTCTA 1106 558 GTCCTCCGGTATGTTCTAA 1107 559 TCCTCCGGTATGTTCTAAA 1108 560 CCTCCGGTATGTTCTAAAG 1109 561 TCCGGTATGTTCTAAAGCT 1111 562 CCATCACTTTGGCTAGTGA 1151 563 # of Starting Nucleotide with ATP1B2 Target reference to Sequence SEQ ID NO: 131 SEQ ID NO: CCGAGGACGCACCAGTTTA 653 564 CGAGGACGCACCAGTTTAT 654 565 TGCAGACTGTCTCCGACCA 771 566 CAGACTGTCTCCGACCATA 773 567 CAAGACTGAGAACCTTGAT 841 568 AGAACCTTGATGTCATTGT 849 569 CCTTGATGTCATTGTCAAT 853 570 AAGTTCTTGGAGCCTTACA 917 571 AGTTCTTGGAGCCTTACAA 918 572 GAGCCTTACAACGACTCTA 926 573 AGCCTTACAACGACTCTAT 927 574 TTACAACGACTCTATCCAA 931 575 GCTATTACGAACAGCCAGA 981 576 TATTACGAACAGCCAGATA 983 577 ATTACGAACAGCCAGATAA 984 578 CAGATAATGGAGTCCTCAA 996 579 GATAATGGAGTCCTCAACT 998 580 AAACGTGCCTGCCAATTCA 1022 581 AACGTGCCTGCCAATTCAA 1023 582 AACCAGAGCATGAATGTTA 1160 583 CTCGGCAACTTCGTCATGT 1214 584 AATGTAGAATGTCGCATCA 1355 585 ATGTAGAATGTCGCATCAA 1356 586 CAACATCGCCACAGACGAT 1381 587 GACGATGAGCGAGACAAGT 1394 588 TGGCCTTCAAACTCCGCAT 1425 589 CCATCTCTCTCCTGTGGAT 1474 590 TTTGATAACAGAGCTATGA 1550 591 CCATTGCGGTTCCGTCACT 1620 592 AGGAGTTAGGAGCCTTTCT 1707 593 TGTGAGAGCTATCCACTCT 1740 594 CACTCTCCTGCCTGCATAT 1753 595 CGCCACACACACACACAAA 1825 596 TCTACACAGTCGCCATCTT 1956 597 TCGCCATCTTGGTGACTTT 1965 598 GGTTGACCTAGGCTGAATA 2598 599 GTTGACCTAGGCTGAATAT 2599 600 GGCTGAATATCCACTTTGT 2608 601 AGCAAGTTATCAACTAATC 2828 602 GCAAGTTATCAACTAATCA 2829 603 CCAAATCTAGCCTCTGAAT 2888 604 CTCCTGCTCTGAATATTCT 3012 605 TGTGTCAGATCTACTGTAA 3251 606 # of Starting Nucleotide with ATP1B3 Target reference to Sequence SEQ ID NO: 132 SEQ ID NO: TTGCTCTTCTACCTAGTTT 292 607 CAGTGACCGCATTGGAATA 434 608 GACCGCATTGGAATATACA 438 609 TTCAGTAGGTCTGATCCAA 457 610 CAGTAGGTCTGATCCAACT 459 611 GGTACATTGAAGACCTTAA 488 612 TACATTGAAGACCTTAAGA 490 613 AGACCTTAAGAAGTTTCTA 498 614 GACCTTAAGAAGTTTCTAA 499 615 GTTTATGTTGCATGTCAGT 592 616 TGGTATGAATGATCCTGAT 639 617 TGAAGGAGTGCCAAGGATA 723 618 TGTAGCAGTTTATCCTCAT 774 619 GTAGCAGTTTATCCTCATA 775 620 CTCATAATGGAATGATAGA 788 621 AGCCATTGGTTGCTGTTCA 857 622 GCCATTGGTTGCTGTTCAG 858 623 GTAACAGTTGAGTGCAAGA 910 624 TAACAGTTGAGTGCAAGAT 911 625 TGATGGATCAGCCAACCTA 930 626 GATGGATCAGCCAACCTAA 931 627 ATGGATCAGCCAACCTAAA 932 628 GCATAGTATGAGTAGGATA 1009 629 CATAGTATGAGTAGGATAT 1010 630 GGATATCTCCACAGAGTAA 1023 631 GATATCTCCACAGAGTAAA 1024 632 AGAAAGGTGTGTGGTACAT 1111 633 ATAACGTGCTTCCAGATCA 1146 634 TAACGTGCTTCCAGATCAT 1147 635 AGTGTACAGTCGCCAGATA 1220 636 GTGAACACCTGATTCCAAA 1246 637 AGCTTAATATGCCGTGCTA 1321 638 TAATATGCCGTGCTATGTA 1325 639 AATATGCCGTGCTATGTAA 1326 640 ATATGCCGTGCTATGTAAA 1327 641 GCCGTGCTATGTAAATATT 1331 642 TGCAAGAAATGTGGTATGT 1437 643 ATGCTGAATTAGCCTCGAT 1548 644 TTGATTAAGAGCACAAACT 1571 645 AGCAGACTGTGGACTGTAA 1785 646 GCAGACTGTGGACTGTAAT 1786 647 CAGACTGTGGACTGTAATA 1787 648

Table 5 lists examples of siRNA target sequences within the SLC12A1 and SLC12A2 DNA sequences (SEQ ID NO:7 and SEQ ID NO:133, respectively) from which siRNAs of the present invention are designed in a manner as set forth above. As noted above, SLC12A1 and SLC12A2 encode the Na—K-2Cl cotransporter, NKCC2 and NKCC1, respectively.

TABLE 5 SLC12A1 Target Sequences for siRNAs # of Starting Nucleotide with SLC12A1 Target reference to Sequence SEQ ID NO: 7 SEQ ID NO: CCACCATAGTAACGACAAT 675 73 GGAATGGAATGGGAGGCAA 974 74 GGGATGAACTGCAATGGTT 1373 75 CCATGCCTCTTATGCCAAA 1780 76 CCTGCTCTCCTGGACATAA 2102 77 GCATCTGCTGTGAAGTCTT 2151 78 GCCTCAGGCTTAGGAAGAA 2315 79 GGAAGCGACTATCAAAGAT 2542 80 GCTGGCAAGTTGAACATTA 2609 81 GCAAGAAAGGGATCCATAT 3197 82 TAATACCAATCGCTTTCAA 67 649 ACCAATCGCTTTCAAGTTA 71 650 CAATCGCTTTCAAGTTAGT 73 651 ATAGAGTACTATCGTAACA 353 652 CCAGCCTGCTTGAGATTCA 405 653 CTGTAGTAGATCTACTTAA 864 654 ACCAATGACATCCGGATTA 911 655 CCAATGACATCCGGATTAT 912 656 CAATGACATCCGGATTATA 913 657 GGCTATGACTTCTCAAGAT 1409 658 GCCTCATATGCACTTATTA 1748 659 AGACCTGCGTATGGAATTT 1811 660 ACGTCTATGTGACTTGTAA 1935 661 GTCTATGTGACTTGTAAGA 1937 662 TTCCTACGTGAGTGCTTTA 1993 663 GACAATGCTCTGGAATTAA 2012 664 CTCTGGTGATTGGATATAA 2346 665 TGACAGAGATTGAGAACTA 2388 666 TGAGATTGGCGTGGTTATA 2437 667 GCATCCGAGGCTTGTTTAA 2586 668 ACCATATCGTCTCCATGAA 3007 669 CCATATCGTCTCCATGAAA 3008 670 TGAAAGCTGCAAAGATTTA 3022 671 TCGACTGAATGAACTCTTA 3130 672 CCATATCGGATTTGTTGTA 3210 673 GGTTGGAAATCCTCACAAA 3237 674 CTTACTAGTTAGAGGAAAT 3271 675 # of Starting Nucleotide with SLC12A2 Target reference to Sequence SEQ ID NO: 133 SEQ ID NO: ACCACCAGCACTACTATTA 748 676 CCACCAGCACTACTATTAT 749 677 CAGCACTACTATTATGATA 753 678 CTATCAGTCCTTGTAATAA 1119 679 ATTGTCTACTTCAGCAATA 1169 680 TATTGGTGATTTCGTCATA 1499 681 TTCGTCATAGGAACATTTA 1509 682 TAATGACACTATCGTAACA 1820 683 GATGTTTGCTAAAGGTTAT 2081 684 CTTCGTGGCTACATCTTAA 2118 685 TGCACTTGGATTCATCTTA 2147 686 GATGATCTGTGGCCATGTA 2615 687 CTCGAAGACAAGCCATGAA 2644 688 TGAAAGAGATGTCCATCGA 2659 689 AGAGATGTCCATCGATCAA 2663 690 CCATCGATCAAGCCAAATA 2671 691 CATCGATCAAGCCAAATAT 2672 692 GGTCGTATGAAGCCAAACA 2793 693 CACTTGTCCTTGGATTTAA 2812 694 TAGTGGTTATTCGCCTAAA 2914 695 ATCTCATCTTCAAGGACAA 2948 696 CGATTTAGATACTTCCAAA 3044 697 TCATTGGTGGAAAGATAAA 3334 698 TTAGCAAGTTCCGGATAGA 3391 699 GAAATCATTGAGCCATACA 3480 700 AGCAAGATATTGCAGATAA 3520 701 GATGAACCATGGCGAATAA 3549 702 CATTCAAGCACAGCTAATA 3639 703 TTCAGTGCCTAGTGTAGTA 3840 704 AGGAAAGTTGCTCCATTGA 3941 705 AAAGTTGCTCCATTGATAA 3944 706 CAATCTTAATGGTGATTCT 4001 707 TTGACATCATAGTCTAGTA 4995 708 GACATCATAGTCTAGTAAA 4997 709 GTGTGTGTGTGTGTATATA 5141 710 GTGTGTGTGTGTATATATA 5143 711 TAGGCAAACTTTGGTTTAA 5249 712 GGAGAATACTTCGCCTAAA 5375 713 TGAGTATGACCTAGGTATA 5834 714 AGAGATCTGATAACTTGAA 5852 715 GGTAAAGACAGTAGAAATA 5981 716 TTTAAGCTCTGGTGGATGA 6678 717 As cited in the examples above, one of skill in the art is able to use the target sequence information provided in Tables 1-5 to design interfering RNAs having a length shorter or longer than the sequences provided in Table 1-5 by referring to the sequence position in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, and adding or deleting nucleotides complementary or near complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, respectively.

The target RNA cleavage reaction guided by siRNAs and other forms of interfering RNA is highly sequence specific. In general, siRNA containing a sense nucleotide strand identical in sequence to a portion of the target mRNA and an antisense nucleotide strand exactly complementary to a portion of the target mRNA are siRNA embodiments for inhibition of mRNAs cited herein. However, 100% sequence complementarity between the antisense siRNA strand and the target mRNA, or between the antisense siRNA strand and the sense siRNA strand, is not required to practice the present invention. Thus, for example, the invention allows for sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.

In one embodiment of the invention, the antisense strand of the siRNA has at least near-perfect contiguous complementarity of at least 19 nucleotides with the target mRNA. “Near-perfect,” as used herein, means the antisense strand of the siRNA is “substantially complementary to,” and the sense strand of the siRNA is “substantially identical” to at least a portion of the target mRNA. “Identity,” as known by one of ordinary skill in the art, is the degree of sequence relatedness between nucleotide sequences as determined by matching the order and identity of nucleotides between the sequences. In one embodiment, the antisense strand of an siRNA having 80% and between 80% up to 100% complementarity, for example, 85%, 90% or 95% complementarity, to the target mRNA sequence are considered near-perfect complementarity and may be used in the present invention. “Perfect” contiguous complementarity is standard Watson-Crick base pairing of adjacent base pairs. “At least near-perfect” contiguous complementarity includes “perfect” complementarity as used herein. Computer methods for determining identity or complementarity are designed to identify the greatest degree of matching of nucleotide sequences, for example, BLASTN (Altschul, S. F., et al. (1990) J. Mol. Biol. 215:403-410).

The term “percent identity” describes the percentage of contiguous nucleotides in a first nucleic acid molecule that is the same as in a set of contiguous nucleotides of the same length in a second nucleic acid molecule. The term “percent complementarity” describes the percentage of contiguous nucleotides in a first nucleic acid molecule that can base pair in the Watson-Crick sense with a set of contiguous nucleotides in a second nucleic acid molecule.

The relationship between a target mRNA (sense strand) and one strand of an siRNA (the sense strand) is that of identity. The sense strand of an siRNA is also called a passenger strand, if present. The relationship between a target mRNA (sense strand) and the other strand of an siRNA (the antisense strand) is that of complementarity. The antisense strand of an siRNA is also called a guide strand.

The penultimate base in a nucleic acid sequence that is written in a 5′ to 3′ direction is the next to the last base, i.e., the base next to the 3′ base. The penultimate 13 bases of a nucleic acid sequence written in a 5′ to 3′ direction are the last 13 bases of a sequence next to the 3′ base and not including the 3′ base. Similarly, the penultimate 14, 15, 16, 17, or 18 bases of a nucleic acid sequence written in a 5′ to 3′ direction are the last 14, 15, 16, 17, or 18 bases of a sequence, respectively, next to the 3′ base and not including the 3′ base.

The phrase “a region of at least 13 contiguous nucleotides having at least 90% sequence complementarity to, or at least 90% sequence identity with, the penultimate 13 nucleotides of the 3′ end of any one of (a sequence identifier)” allows a one nucleotide substitution. Two nucleotide substitutions (i.e., 11/13=85% identity/complementarity) are not included in such a phrase.

In one embodiment of the invention, the region of contiguous nucleotides is a region of at least 14 contiguous nucleotides having at least 85% sequence complementarity to, or at least 85% sequence identity with, the penultimate 14 nucleotides of the 3′ end of the sequence identified by each sequence identifier. Two nucleotide substitutions (i.e., 12/14=86% identity/complementarity) are included in such a phrase.

In a further embodiment of the invention, the region of contiguous nucleotides is a region of at least 15, 16, 17, or 18 contiguous nucleotides having at least 80% sequence complementarity to, or at least 80% sequence identity with, the penultimate 14 nucleotides of the 3′ end of the sequence of the sequence identifier. Three nucleotide substitutions are included in such a phrase.

The target sequence in the mRNAs corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, may be in the 5′ or 3′ untranslated regions of the mRNA as well as in the coding region of the mRNA.

One or both of the strands of double-stranded interfering RNA may have a 3′ overhang of from 1 to 6 nucleotides, which may be ribonucleotides or deoxyribonucleotides or a mixture thereof. The nucleotides of the overhang are not base-paired. In one embodiment of the invention, the interfering RNA comprises a 3′ overhang of TT or UU. In another embodiment of the invention, the interfering RNA comprises at least one blunt end. The termini usually have a 5′ phosphate group or a 3′ hydroxyl group. In other embodiments, the antisense strand has a 5′ phosphate group, and the sense strand has a 5′ hydroxyl group. In still other embodiments, the termini are further modified by covalent addition of other molecules or functional groups.

The sense and antisense strands of the double-stranded siRNA may be in a duplex formation of two single strands as described above or may be a single molecule where the regions of complementarity are base-paired and are covalently linked by a hairpin loop so as to form a single strand. It is believed that the hairpin is cleaved intracellularly by a protein termed dicer to form an interfering RNA of two individual base-paired RNA molecules.

Interfering RNAs may differ from naturally-occurring RNA by the addition, deletion, substitution or modification of one or more nucleotides. Non-nucleotide material may be bound to the interfering RNA, either at the 5′ end, the 3′ end, or internally. Such modifications are commonly designed to increase the nuclease resistance of the interfering RNAs, to improve cellular uptake, to enhance cellular targeting, to assist in tracing the interfering RNA, to further improve stability, or to reduce the potential for activation of the interferon pathway. For example, interfering RNAs may comprise a purine nucleotide at the ends of overhangs. Conjugation of cholesterol to the 3′ end of the sense strand of an siRNA molecule by means of a pyrrolidine linker, for example, also provides stability to an siRNA.

Further modifications include a 3′ terminal biotin molecule, a peptide known to have cell-penetrating properties, a nanoparticle, a peptidomimetic, a fluorescent dye, or a dendrimer, for example.

Nucleotides may be modified on their base portion, on their sugar portion, or on the phosphate portion of the molecule and function in embodiments of the present invention. Modifications include substitutions with alkyl, alkoxy, amino, deaza, halo, hydroxyl, thiol groups, or a combination thereof, for example. Nucleotides may be substituted with analogs with greater stability such as replacing a ribonucleotide with a deoxyribonucleotide, or having sugar modifications such as 2′ OH groups replaced by 2′ amino groups, 2′ O-methyl groups, 2′ methoxyethyl groups, or a 2′-O, 4′-C methylene bridge, for example. Examples of a purine or pyrimidine analog of nucleotides include a xanthine, a hypoxanthine, an azapurine, a methylthioadenine, 7-deaza-adenosine and O- and N-modified nucleotides. The phosphate group of the nucleotide may be modified by substituting one or more of the oxygens of the phosphate group with nitrogen or with sulfur (phosphorothioates). Modifications are useful, for example, to enhance function, to improve stability or permeability, or to direct localization or targeting.

There may be a region or regions of the antisense interfering RNA strand that is (are) not complementary to a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134. Non-complementary regions may be at the 3′, 5′ or both ends of a complementary region or between two complementary regions.

Interfering RNAs may be generated exogenously by chemical synthesis, by in vitro transcription, or by cleavage of longer double-stranded RNA with dicer or another appropriate nuclease with similar activity. Chemically synthesized interfering RNAs, produced from protected ribonucleoside phosphoramidites using a conventional DNA/RNA synthesizer, may be obtained from commercial suppliers such as Ambion Inc. (Austin, Tex.), Invitrogen (Carlsbad, Calif.), or Dharmacon (Lafayette, Colo.). Interfering RNAs are purified by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination thereof, for example. Alternatively, interfering RNA may be used with little if any purification to avoid losses due to sample processing.

Interfering RNAs can also be expressed endogenously from plasmid or viral expression vectors or from minimal expression cassettes, for example, PCR generated fragments comprising one or more promoters and an appropriate template or templates for the interfering RNA. Examples of commercially available plasmid-based expression vectors for shRNA include members of the pSilencer series (Ambion, Austin, Tex.) and pCpG-siRNA (InvivoGen, San Diego, Calif.). Viral vectors for expression of interfering RNA may be derived from a variety of viruses including adenovirus, adeno-associated virus, lentivirus (e.g., HIV, FIV, and EIAV), and herpes virus. Examples of commercially available viral vectors for shRNA expression include pSilencer adeno (Ambion, Austin, Tex.) and pLenti6/BLOCK-iT™-DEST (Invitrogen, Carlsbad, Calif.). Selection of viral vectors, methods for expressing the interfering RNA from the vector and methods of delivering the viral vector are within the ordinary skill of one in the art. Examples of kits for production of PCR-generated shRNA expression cassettes include Silencer Express (Ambion, Austin, Tex.) and siXpress (Mirus, Madison, Wis.).

Interfering RNAs may be expressed from a variety of eukaryotic promoters known to those of ordinary skill in the art, including pol III promoters, such as the U6 or H1 promoters, or pol II promoters, such as the cytomegalovirus promoter. Those of skill in the art will recognize that these promoters can also be adapted to allow inducible expression of the interfering RNA.

Hybridization under Physiological Conditions: In certain embodiments of the present invention, an antisense strand of an interfering RNA hybridizes with an mRNA in vivo as part of the RISC complex.

“Hybridization” refers to a process in which single-stranded nucleic acids with complementary or near-complementary base sequences interact to form hydrogen-bonded complexes called hybrids. Hybridization reactions are sensitive and selective. In vitro, the specificity of hybridization (i.e., stringency) is controlled by the concentrations of salt or formamide in prehybridization and hybridization solutions, for example, and by the hybridization temperature; such procedures are well known in the art. In particular, stringency is increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.

For example, high stringency conditions could occur at about 50% formamide at 37° C. to 42° C. Reduced stringency conditions could occur at about 35% to 25% formamide at 30° C. to 35° C. Examples of stringency conditions for hybridization are provided in Sambrook, J., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Further examples of stringent hybridization conditions include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing, or hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in 0.3×SSC, or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. The temperature for hybridization is about 5-10° C. less than the melting temperature (T_(m)) of the hybrid where T_(m) is determined for hybrids between 19 and 49 base pairs in length using the following calculation: T_(m)° C.=81.5+16.6(log₁₀[Na+])+0.41 (% G+C)− (600/N) where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer.

The above-described in vitro hybridization assay provides a method of predicting whether binding between a candidate siRNA and a target will have specificity. However, in the context of the RISC complex, specific cleavage of a target can also occur with an antisense strand that does not demonstrate high stringency for hybridization in vitro.

Single-stranded interfering RNA: As cited above, interfering RNAs ultimately function as single strands. Single-stranded (ss) interfering RNA has been found to effect mRNA silencing, albeit less efficiently than double-stranded RNA. Therefore, embodiments of the present invention also provide for administration of a ss interfering RNA that hybridizes under physiological conditions to a portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:101, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, or SEQ ID NO:134, respectively. The ss interfering RNA has a length of 19 to 49 nucleotides as for the ds interfering RNA cited above. The ss interfering RNA has a 5′ phosphate or is phosphorylated in situ or in vivo at the 5′ position. The term “5′ phosphorylated” is used to describe, for example, polynucleotides or oligonucleotides having a phosphate group attached via ester linkage to the C5 hydroxyl of the sugar (e.g., ribose, deoxyribose, or an analog of same) at the 5′ end of the polynucleotide or oligonucleotide.

SS interfering RNAs are synthesized chemically or by in vitro transcription or expressed endogenously from vectors or expression cassettes as for ds interfering RNAs. 5′ Phosphate groups may be added via a kinase, or a 5′ phosphate may be the result of nuclease cleavage of an RNA. Delivery is as for ds interfering RNAs. In one embodiment, ss interfering RNAs having protected ends and nuclease resistant modifications are administered for silencing. SS interfering RNAs may be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to inhibit annealing or for stabilization.

Hairpin interfering RNA: A hairpin interfering RNA is a single molecule (e.g., a single oligonucleotide chain) that comprises both the sense and antisense strands of an interfering RNA in a stem-loop or hairpin structure (e.g., a shRNA). For example, shRNAs can be expressed from DNA vectors in which the DNA oligonucleotides encoding a sense interfering RNA strand are linked to the DNA oligonucleotides encoding the reverse complementary antisense interfering RNA strand by a short spacer. If needed for the chosen expression vector, 3′ terminal T's and nucleotides forming restriction sites may be added. The resulting RNA transcript folds back onto itself to form a stem-loop structure.

Mode of administration: Interfering RNA may be delivered directly to the eye by ocular tissue injection such as periocular, conjunctival, subtenon, intracameral, intravitreal, intraocular, subretinal, subconjunctival, retrobulbar, or intracanalicular injections; by direct application to the eye using a catheter or other placement device such as a retinal pellet, intraocular insert, suppository or an implant comprising a porous, non-porous, or gelatinous material; by topical ocular drops or ointments; or by a slow release device in the cul-de-sac or implanted adjacent to the sclera (transscleral) or within the eye. Intracameral injection may be through the cornea into the anterior chamber to allow the agent to reach the trabecular meshwork. Intracanalicular injection may be into the venous collector channels draining Schlemm's canal or into Schlemm's canal. Systemic or parenteral administration is contemplated including but not limited to intravenous, subcutaneous, and oral delivery.

Subject: A subject in need of treatment for ocular hypertension or at risk for developing ocular hypertension is a human or other mammal having ocular hypertension or at risk of having ocular hypertension associated with undesired or inappropriate expression or activity of targets as cited herein, i.e., carbonic anhydrase II, IV, or XII; β1- or β2-adrenergic receptors; acetylcholinesterase; Na⁺/K⁺-ATPase; or Na—K-2Cl cotransporter. Ocular structures associated with such disorders may include the eye, retina, choroid, lens, cornea, trabecular meshwork, iris, optic nerve, optic nerve head, sclera, aqueous chamber, vitreous chamber, or ciliary body, for example. A subject may also be an ocular cell, cell culture, organ or an ex vivo organ or tissue.

Formulations and Dosage: Pharmaceutical formulations comprise an interfering RNA, or salt thereof, of the invention up to 99% by weight mixed with a physiologically acceptable ophthalmic carrier medium such as water, buffer, saline, glycine, hyaluronic acid, mannitol, and the like.

Interfering RNAs of the present invention are administered as solutions, suspensions, or emulsions. The following are examples of possible formulations embodied by this invention.

Amount in weight % Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0 Hydroxypropylmethylcellulose 0.5 Sodium chloride 0.8 Benzalkonium Chloride 0.01 EDTA 0.01 NaOH/HCl qs pH 7.4 Purified water (RNase-free) qs 100 mL Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0 Phosphate Buffered Saline 1.0 Benzalkonium Chloride 0.01 Polysorbate 80 0.5 Purified water (RNase-free) q.s. to 100% Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0 Monobasic sodium phosphate 0.05 Dibasic sodium phosphate 0.15 (anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05 Cremophor EL 0.1 Benzalkonium chloride 0.01 HCl and/or NaOH pH 7.3-7.4 Purified water (RNase-free) q.s. to 100% Interfering RNA up to 99; 0.1-99; 0.1-50; 0.5-10.0 Phosphate Buffered Saline 1.0 Hydroxypropyl-β-cyclodextrin 4.0 Purified water (RNase-free) q.s. to 100%

Generally, an effective amount of the interfering RNA of embodiments of the invention results in an extracellular concentration at the surface of the target cell of from 100 pM to 100 nM, or from 1 nM to 50 nM, or from 5 nM to about 10 nM, or to about 25 nM. The dose required to achieve this local concentration will vary depending on a number of factors including the delivery method, the site of delivery, the number of cell layers between the delivery site and the target cell or tissue, whether delivery is local or systemic, etc. The concentration at the delivery site may be considerably higher than it is at the surface of the target cell or tissue. Topical compositions are delivered to the surface of the eye one to four times per day, or on an extended delivery schedule such as daily, weekly, bi-weekly, monthly, or longer, according to the routine discretion of a skilled clinician. The pH of the formulation is about pH 4-9, or pH 4.5 to pH 7.4.

Therapeutic treatment of patients with siRNAs directed against the ocular hypertension target mRNAs is expected to be beneficial over small molecule topical ocular drops by increasing the duration of action, thereby allowing less frequent dosing and greater patient compliance.

While the precise regimen is left to the discretion of the clinician, interfering RNA may be administered by placing one drop in each eye as directed by the clinician. An effective amount of a formulation may depend on factors such as the age, race, and sex of the subject, the severity of the ocular hypertension, the rate of target gene transcript/protein turnover, the interfering RNA potency, and the interfering RNA stability, for example. In one embodiment, the interfering RNA is delivered topically to the eye and reaches the trabecular meshwork, retina or optic nerve head at a therapeutic dose thereby ameliorating an ocular hypertension-associated disease process.

Acceptable carriers: An ophthalmically acceptable carrier refers to those carriers that cause at most, little to no ocular irritation, provide suitable preservation if needed, and deliver one or more interfering RNAs of the present invention in a homogenous dosage. An acceptable carrier for administration of interfering RNA of embodiments of the present invention include the cationic lipid-based transfection reagents TransIT®-TKO (Mirus Corporation, Madison, Wis.), LIPOFECTIN®, Lipofectamine, OLIGOFECTAMINE™ (Invitrogen, Carlsbad, Calif.), or DHARMAFECT™ (Dharmacon, Lafayette, Colo.); polycations such as polyethyleneimine; cationic peptides such as Tat, polyarginine, or Penetratin (Antp peptide); or liposomes. Liposomes are formed from standard vesicle-forming lipids and a sterol, such as cholesterol, and may include a targeting molecule such as a monoclonal antibody having binding affinity for endothelial cell surface antigens, for example. Further, the liposomes may be PEGylated liposomes.

The interfering RNAs may be delivered in solution, in suspension, or in bioerodible or non-bioerodible delivery devices. The interfering RNAs can be delivered alone, as components of covalent conjugates, complexed with cationic lipids, cationic peptides, or cationic polymers, or encapsulated in targeted or non-targeted nanoparticles.

For ophthalmic delivery, an interfering RNA may be combined with ophthalmologically acceptable preservatives, co-solvents, surfactants, viscosity enhancers, penetration enhancers, buffers, sodium chloride, or water to form an aqueous, sterile ophthalmic suspension or solution. Ophthalmic solution formulations may be prepared by dissolving the interfering RNA in a physiologically acceptable isotonic aqueous buffer. Further, the ophthalmic solution may include an ophthalmologically acceptable surfactant to assist in dissolving the inhibitor. Viscosity building agents, such as hydroxymethyl cellulose, hydroxyethyl cellulose, methylcellulose, polyvinylpyrrolidone, or the like may be added to the compositions of the present invention to improve the retention of the compound.

In order to prepare a sterile ophthalmic ointment formulation, the interfering RNA is combined with a preservative in an appropriate vehicle, such as mineral oil, liquid lanolin, or white petrolatum. Sterile ophthalmic gel formulations may be prepared by suspending the interfering RNA in a hydrophilic base prepared from the combination of, for example, CARBOPOL®-940 (BF Goodrich, Charlotte, N.C.), or the like, according to methods known in the art for other ophthalmic formulations. VISCOAT® (Alcon Laboratories, Inc., Fort Worth, Tex.) may be used for intraocular injection, for example. Other compositions of the present invention may contain penetration enhancing agents such as cremephor and TWEEN® 80 (polyoxyethylene sorbitan monolaurate, Sigma Aldrich, St. Louis, Mo.), in the event the interfering RNA is less penetrating in the eye.

Kits: Embodiments of the present invention provide a kit that includes reagents for attenuating the expression of an mRNA as cited herein in a cell. The kit contains an siRNA or an shRNA expression vector. For siRNAs and non-viral shRNA expression vectors the kit also may contain a transfection reagent or other suitable delivery vehicle. For viral shRNA expression vectors, the kit may contain the viral vector and/or the necessary components for viral vector production (e.g., a packaging cell line as well as a vector comprising the viral vector template and additional helper vectors for packaging). The kit may also contain positive and negative control siRNAs or shRNA expression vectors (e.g., a non-targeting control siRNA or an siRNA that targets an unrelated mRNA). The kit also may contain reagents for assessing knockdown of the intended target gene (e.g., primers and probes for quantitative PCR to detect the target mRNA and/or antibodies against the corresponding protein for western blots). Alternatively, the kit may comprise an siRNA sequence or an shRNA sequence and the instructions and materials necessary to generate the siRNA by in vitro transcription or to construct an shRNA expression vector.

A pharmaceutical combination in kit form is further provided that includes, in packaged combination, a carrier means adapted to receive a container means in close confinement therewith and a first container means including an interfering RNA composition and an ophthalmically acceptable carrier. Such kits can further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Printed instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, can also be included in the kit.

The ability of interfering RNA to knock-down the levels of endogenous target gene expression in, for example, human trabecular meshwork (TM) cells is evaluated in vitro as follows. Transformed human TM cells, for example, cell lines designated GTM-3 or HTM-3 (see Pang, I. H. et al., 1994. Curr. Eye Res. 13:51-63), are plated 24 h prior to transfection in standard growth medium (e.g., DMEM supplemented with 10% fetal bovine serum). Transfection is performed using Dharmafect 1 (Dharmacon, Lafayette, Colo.) according to the manufacturer's instructions at interfering RNA concentrations ranging from 0.1 nM-100 nM. Non-targeting control interfering RNA and lamin A/C interfering RNA (Dharmacon) are used as controls. Target mRNA levels are assessed by qPCR 24 h post-transfection using, for example, TAQMAN® forward and reverse primers and a probe set that encompasses the target site (Applied Biosystems, Foster City, Calif.). Target protein levels may be assessed approximately 72 h post-transfection (actual time dependent on protein turnover rate) by western blot, for example. Standard techniques for RNA and/or protein isolation from cultured cells are well-known to those skilled in the art. To reduce the chance of non-specific, off-target effects, the lowest possible concentration of interfering RNA should be used that will produce the desired level of knock-down in target gene expression.

The ability of interfering RNAs of the present invention to knock-down levels of CA2 protein expression is further exemplified in Example 1 as follows.

Example 1 Interfering RNA for Specifically Silencing CA2 in HeLa Cells

The present study examines the ability of CA2-interfering RNA to knock down the levels of endogenous CA2 expression in cultured HeLa cells.

Transfection of HeLa cells was accomplished using standard in vitro concentrations (100 nM and 1 nM) of CA2 siRNAs, or a non-targeting control siRNA and DharmaFECT™ 1 transfection reagent (Dharmacon, Lafayette, Colo.). All siRNAs were dissolved in 1×siRNA buffer, an aqueous solution of 20 mM KCl, 6 mM HEPES (pH 7.5), 0.2 mM MgCl₂. CA2 protein expression and actin protein expression (loading control) was evaluated by western blot analysis 72 hours post-transfection. The CA2 siRNAs are double-stranded interfering RNAs having specificity for the following target sequences: siCA2#1 targets SEQ ID NO:721; siCA2#3 targets SEQ ID NO:15; siCA2#4 targets SEQ ID NO:720; siCA2#5 targets SEQ ID NO:141. Each of the four CA2 siRNAs decreased CA2 expression significantly at both 100 nM and 1 nM relative to a non-targeting control siRNA as shown by the western blot data of FIG. 1. SiCA2#4 targeting SEQ ID NO:720 and siCA2#5 targeting SEQ ID NO:141 appeared to be particularly effective.

The references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated by reference.

Those of skill in the art, in light of the present disclosure, will appreciate that obvious modifications of the embodiments disclosed herein can be made without departing from the spirit and scope of the invention. All of the embodiments disclosed herein can be made and executed without undue experimentation in light of the present disclosure. The full scope of the invention is set out in the disclosure and equivalent embodiments thereof. The specification should not be construed to unduly narrow the full scope of protection to which the present invention is entitled.

As used herein and unless otherwise indicated, the terms “a” and “an” are taken to mean “one”, “at least one” or “one or more”. 

1. A method of attenuating expression of an ocular hypertension target mRNA of a subject, comprising: administering to the subject a composition comprising an effective amount of interfering RNA having a length of 19 to 49 nucleotides and a pharmaceutically acceptable carrier, the interfering RNA comprising: a sense nucleotide strand, an antisense nucleotide strand, and a region of at least near-perfect contiguous complementarity of at least 19 nucleotides; wherein the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, or SEQ ID NO:133, and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, or SEQ ID NO:133, respectively, wherein the expression of an ocular hypertension target mRNA is attenuated.
 2. The method of claim 1 wherein the subject is a human and the human has ocular hypertension.
 3. The method of claim 1 wherein the subject is a human and the human is at risk of developing ocular hypertension.
 4. The method of claim 1 wherein the composition is administered via a topical, intravitreal, transcleral, periocular, conjunctival, subtenon, intracameral, subretinal, subconjunctival, retrobulbar, or intracanalicular route.
 5. The method of claim 1 wherein the composition is administered via in vivo expression from an expression vector capable of expressing the interfering RNA.
 6. The method of claim 1 wherein the ocular hypertension target mRNA encodes a β1- or β2-adrenergic receptor, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:3 or SEQ ID NO:4 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:3 or SEQ ID NO:4, respectively.
 7. The method of claim 1 wherein the ocular hypertension target mRNA encodes an acetylcholinesterase, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:5 or SEQ ID NO:123 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:5 or SEQ ID NO:123, respectively.
 8. The method of claim 1 wherein the ocular hypertension target mRNA encodes a subunit of Na⁺/K⁺-ATPase, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:6, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, or SEQ ID NO:132 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:6, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, or SEQ ID NO:132, respectively.
 9. The method of claim 1 wherein the ocular hypertension target mRNA encodes a Na—K-2Cl cotransporter, and the antisense strand hybridizes under physiological conditions to a portion of mRNA corresponding to SEQ ID NO:7 or SEQ ID NO:133 and has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the hybridizing portion of mRNA corresponding to SEQ ID NO:7 or SEQ ID NO:133, respectively.
 10. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:3 comprising nucleotide 468, 523, 799, 1563, 1565, 1569, 1593, 1613, 1614, 1626, 310, 322, 726, 769, 772, 801, 802, 1501, 1576, 1577, 1579, 1580, 1581, 1586, 1590, 1592, 1594, 1615, 1616, 1632, 1633, or
 1654. 11. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:4 comprising nucleotide 329, 375, 1031, 1046, 1149, 1163, 1371, 1401, 1426, 1880, 283, 607, 608, 609, 619, 623, 722, 857, 1037, 1091, 1115, 1124, 1136, 1137, 1151, 1164, 1393, 1394, 1395, 1406, 1407, 1427, 1428, 1429, 1442, 1725, 1726, 1756, 1757, 1758, 1767, 1790, 1791, 1792, 1793, 1803, 1861, 1869, 1971, 1972, or
 1979. 12. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:123 comprising nucleotide 1875, 1890, 1891, 2011, 2012, 2133, or
 2134. 13. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:5 comprising nucleotide 366, 370, 384, 385, 525, 588, 768, 1045, 1046, 1061, 1090, 1232, 1314, 1316, 1460, 1461, 1462, 1528, 1607, 1705, 1713, 382, 393, 397, 622, 1131, 1459, 1530, 2251, 2885, 2886, 386, 1231, 1315, 2047, 2049, 2053, 2055, 2057, 2125, 2126, 2127, 2250, 2253, 2258, 2260, 2318, 2395, 2397, 2404, 2405, 2643, 2645, or
 2887. 14. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:124 comprising nucleotide 2208, 2275, 2307, 2526, 2538, 2592, 2628, 2979, 2985, 3093, 3474, 3504, 3505, 3506, 3518, 343, 442, 700, 707, 811, 907, 1059, 1363, 1594, 1662, 1758, 1760, 1896, 2037, or
 2147. 15. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:125 comprising nucleotide 436, 441, 443, 552, 617, 701, 702, 832, 2204, 2291, or
 2495. 16. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:6 comprising nucleotide 471, 1990, 3080, 3797, 4037, 4093, 4225, 4323, 5213, 5285, 214, 467, 470, 472, 473, 632, 825, 946, 1693, 1767, 1768, 2157, 2263, 2589, 2590, 2765, 2988, 3094, 3144, 3145, 3344, 3345, 3418, 3666, 3828, 3850, 4040, 4041, 4061, 4882, 4894, 4900, 5040, 5114, 5115, 5128, 5129, 5253, 5296, 5375, 5384, or
 5385. 17. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:126 comprising nucleotide 240, 272, 362, 1836, 1851, 2103, 2137, 2138, 2139, 2157, 2158, 2160, 2425, 2580, 2601, 2646, 2650, 2794, 2803, 3116, 3124, 3126, 3129, or
 3377. 18. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:127 comprising nucleotide 113, 612, 702, 833, 1101, 1732, 1733, 1836, 2070, 2071, 2143, 2328, 2475, 2861, 2862, 2952, 3203, 3281, 3377, 3379, 3470, 3471, 3554, 3614, 3615, 3616, 3617, 3625, 3626, 3642, 3646, 3647, 3653, 3655, 3797, 3801, 3803, 3809 or
 3810. 19. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:128 comprising nucleotide 126, 251, 252, 253, 331, 427, 429, 520, 521, 530, 601, 602, 603, 604, 664, 665, 666, 667, 675, 676, 692, 696, 697, 702, 703, 705, 707, 847, 851, 853, 859, or
 860. 20. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:129 comprising nucleotide 1096, 1099, 1130, 1131, 1167, 1299, 1441, 1450, 1451, 1452, 1564, 1746, 1750, 1751, 1752, 1795, 203, 204, 214, 222, 224, 225, 226, 380, 525, 591, 612, 613, 615, 635, 636, 663, 664, 669, 699, 765, 790, 839, 840, 841, 900, 909, 933, or
 947. 21. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:130 comprising nucleotide 1063, 1102, 1106, 1107, 1108, 1109, 1111, or
 1151. 22. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:131 comprising nucleotide 653, 654, 771, 773, 841, 849, 853, 917, 918, 926, 927, 931, 981, 983, 984, 996, 998, 1022, 1023, 1160, 1214, 1355, 1356, 1381, 1394, 1425, 1474, 1550, 1620, 1707, 1740, 1753, 1825, 1956, 1965, 2598, 2599, 2608, 2828, 2829, 2888, 3012, or
 3251. 23. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:132 comprising nucleotide 292, 434, 438, 457, 459, 488, 490, 498, 499, 592, 639, 723, 774, 775, 788, 857, 858, 910, 911, 930, 931, 932, 1009, 1010, 1023, 1024, 1111, 1146, 1147, 1220, 1246, 1321, 1325, 1326, 1327, 1331, 1437, 1548, 1571, 1785, 1786, or
 1787. 24. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:7 comprising nucleotide 675, 974, 1373, 1780, 2102, 2151, 2315, 2542, 2609, 3197, 67, 71, 73, 353, 405, 864, 911, 912, 913, 1409, 1748, 1811, 1935, 1937, 1993, 2012, 2346, 2388, 2437, 2586, 3007, 3008, 3022, 3130, 3210, 3237, or
 3271. 25. The method of claim 1 wherein the antisense strand is designed to target an mRNA corresponding to SEQ ID NO:133 comprising nucleotide 748, 749, 753, 1119, 1169, 1499, 1509, 1820, 2081, 2118, 2147, 2615, 2644, 2659, 2663, 2671, 2672, 2793, 2812, 2914, 2948, 3044, 3334, 3391, 3480, 3520, 3549, 3639, 3840, 3941, 3944, 4001, 4995, 4997, 5141, 5143, 5249, 5375, 5834, 5852, 5981, or
 6678. 26. The method of claim 1 further comprising administering to the subject a second interfering RNA having a length of 19 to 49 nucleotides, and comprising a sense nucleotide strand, an antisense nucleotide strand, and a region of at least near-perfect complementarity of at least 19 nucleotides; wherein the antisense strand of the second interfering RNA hybridizes under physiological conditions to a second portion of mRNA corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, or SEQ ID NO:133, and the antisense strand has a region of at least near-perfect contiguous complementarity of at least 19 nucleotides with the second hybridizing portion of mRNA corresponding to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, or SEQ ID NO:133, respectively. 